Microrna inhibitors comprising locked nucleotides

ABSTRACT

The invention provides chemically modified oligonucleotides capable of inhibiting the expression (e.g., abundance) of miR-208 family miRNAs, including miR-208a, miR-208b, and/or miR-499. The invention provides in some embodiments, oligonucleotides capable of inhibiting, in a specific fashion, the expression or abundance of each of miR-208a, miR-208b, and miR-499. The invention further provides pharmaceutical compositions comprising the oligonucleotides, and methods of treating patients having conditions or disorders relating to or involving a miR-208 family miRNA, such as a cardiovascular condition. In various embodiments, the oligonucleotides provide advantages in one or more of potency, efficiency of delivery, target specificity, toxicity, and/or stability.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 61/423,456, filed Dec. 15, 2010, and of U.S. ProvisionalApplication No. 61/495,224, filed Jun. 9, 2011, each of which are herebyincorporated by reference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:MIRG_(—)023_(—)01US_SeqList_ST25.txt, date recorded: Dec. 15, 2011, filesize 117 kilobytes).

FIELD OF THE INVENTION

The present invention relates to chemical motifs for microRNA (miRNA ormiR) inhibitors, and particularly to chemically modified miRNA antisenseoligonucleotides having advantages in potency, efficiency of delivery,target specificity, stability, and/or toxicity when administered to apatient.

BACKGROUND

MicroRNAs (miRs) have been implicated in a number of biologicalprocesses including regulation and maintenance of cardiac function (see,Eva Van Rooij and Eric Olson, MicroRNAs: Powerful new regulators ofheart disease and proactive therapeutic targets, J. Clin. Invest.117(9):2369-2376 (2007); Chien K R, Molecular Medicine: MicroRNAs andthe tell-tale heart, Nature 447, 389-390 (2007)). Therefore, miRsrepresent a relatively new class of therapeutic targets for conditionssuch as cardiac hypertrophy, myocardial infarction, heart failure,vascular damage, and pathologic cardiac fibrosis, among others. miRs aresmall, non-protein coding RNAs of about 18 to about 25 nucleotides inlength, and act as repressors of target mRNAs by promoting theirdegradation, when their sequences are perfectly complementary, or byinhibiting translation, when their sequences contain mismatches. Themechanism involves incorporation of the mature miRNA strand into theRNA-induced silencing complex (RISC), where it associates with itstarget RNAs by base-pair complementarity.

miRNA function may be targeted therapeutically by antisensepolynucleotides or by polynucleotides that mimic miRNA function (“miRNAmimetic”). However, targeting miRNAs therapeutically witholigonucleotide-based agents poses several challenges, includingRNA-binding affinity and specificity, efficiency of cellular uptake, andnuclease resistance. For example, when polynucleotides are introducedinto intact cells they are attacked and degraded by nucleases leading toa loss of activity. While polynucleotide analogues have been prepared inan attempt to avoid their degradation, e.g. by means of 2′ substitutions(B. Sproat et al., Nucleic Acids Research 17 (1989), 3373-3386), themodifications often affect the polynucleotide's potency for its intendedbiological action. Such reduced potency, in each case, may be due to aninability of the modified polynucleotide to form a stable duplex withthe target RNA and/or a loss of interaction with the cellular machinery.Other modifications include the use of locked nucleic acid, which hasthe potential to improve RNA-binding affinity. Veedu R N and Wengel J,Locked nucleic acid as a novel class of therapeutic agent. RNA Biology6:3, 321-323 (2009).

Oligonucleotide chemistry patterns or motifs for miRNA inhibitors havethe potential to improve the delivery, stability, potency, specificity,and/or toxicity profile of the inhibitors, and such are needed foreffectively targeting miRNA function in a therapeutic context.

SUMMARY OF THE INVENTION

The invention provides chemically modified oligonucleotides capable ofinhibiting the expression (e.g., abundance) of miR-208 family miRNAs,including miR-208a, miR-208b, and/or miR-499. The invention furtherprovides pharmaceutical compositions comprising the oligonucleotides,and methods of treating patients having conditions or disorders'relating to, or involving, a miR-208 family miRNA. Such conditionsinclude various cardiovascular conditions. In various embodiments, theoligonucleotides provide advantages in one or more of potency;efficiency of delivery, target specificity, toxicity, and/or stability.

In one aspect, the invention provides a chemically-modifiedoligonucleotide capable of reducing the expression or abundance ofmiR-208 family miRNAs. The activity or potency of the oligonucleotidesmay be determined in vitro and/or in vivo. For example, theoligonucleotide may significantly inhibit (e.g., about 50% inhibition)the activity of a miR-208 family miRNA (as determined in the dualluciferase assay) at a concentration of about 50 nM or less, or in otherembodiments, 40 nM or less, 20 nM or less, or 10 nM or less.Alternatively, or in addition, the activity of the oligonucleotide maybe determined in a suitable mouse or rat model, or non-human primatemodel, such as those described herein, where inhibition (e.g., by atleast 50%) of a miR-208 family miRNA is observed at a dose of 50 mg/kgor less, such as 25 mg/kg or less, 10 mg/kg or less, or 5 mg/kg or less.In these embodiments, the oligonucleotide may be dosed subcutaneously orintravenously (and as described herein), and may be formulated in anaqueous preparation (e.g., saline).

The nucleotide sequence of the oligonucleotide is substantiallycomplementary to a nucleotide sequence of human miR-208a or miR-208b (orcorresponding pre- or pri-miRNA), and contains a mixture of locked andnon-locked nucleotides. For example, the oligonucleotide may contain atleast three, at least five, or at least seven, locked nucleotides, andat least one non-locked nucleotide. Generally, the length of theoligonucleotide and number and position of locked nucleotides is suchthat the oligonucleotide reduces miR-208a, miR-208b, and/or mill-499activity at an oligonucleotide concentration of about 50 nM or less inan in vitro luciferase assay, or at a dose of 50 mg/kg or less in asuitable rat or mouse model or non-human primate model as describedherein. In exemplary embodiments, the locked nucleotides have a 2′ to 4′methylene bridge.

The oligonucleotide may comprise, consist essentially of, or consist of,a full length or truncated miR-208a, miR-208b, or miR-499 antisensesequence. In these embodiments, the oligonucleotide is from about 6 to22 nucleotides in length, or is from about 10 to 18 nucleotides inlength, or is about 11 to about 16 nucleotides in length. Theoligonucleotide in some embodiments is about 14, 15, 16, or 17nucleotides in length. The oligonucleotide may comprise the nucleotidesequence of 5′-TGCTCGTCTTA-3′ (SEQ ID NO:1) or may comprise thenucleotide sequence of 5′-TGTTCGTCTTA-3′ (SEQ ID NO:2). In particularembodiments, the oligonucleotide comprises, consists essentially of, orconsists of the nucleotide sequence 5′-CTTTTTGCTCGTCTTA-3′ (SEQ ID NO:3)or 5′-CCTTTTGTTCGTCTTA-3′ (SEQ ID NO:4).

The oligonucleotide may contain at least about 3, at least about 5, orat least about 7 locked nucleotides, or at least 9 locked nucleotides,but in various embodiments is not fully comprised of locked nucleotides.Generally, the number and position of locked nucleotides is such thatthe oligonucleotide reduces or inhibits miR-208a, miR-208b, and/ormiR-499 activity at high potency. In certain embodiments, theoligonucleotide does not contain a stretch of nucleotides with more thanfour, or more than three, or more than two, contiguous non-lockednucleotides. In exemplary embodiments, the oligonucleotide has exactly 9locked nucleotides and 7 non-locked nucleotides. For example, thepattern of locked nucleotides may be such that at least positions 1, 6,10, 13, and 15 are locked nucleotides. In certain embodiments, at leastpositions 1, 5, 10, and 16 are locked nucleotides. In certainembodiments, positions 1, 5, 6, 8, 10, 11, 13, 15, and 16 are lockednucleotides, and the remaining positions are non-locked nucleotides. Inother embodiments, positions 1, 3, 4, 5, 6, 8, 10, 13, 15, and 16 arelocked nucleotides, with the remaining positions being non-lockednucleotides. In still other embodiments, positions 1, 4, 5, 7, 9, 10,12, 14, and 16 are locked nucleotides, with remaining positions beingnon-locked nucleotides. These patterns of locked nucleotides may beemployed, in certain embodiments, using the nucleotide sequence of SEQID NO:3 or SEQ ID NO:4, or variant thereof described herein. Where theinhibitor consists of, or consists essentially of, the nucleotidesequence of SEQ ID NO:3 or SEQ ID NO:4, the oligonucleotide may containall locked nucleotides.

For non-locked nucleotides, the nucleotide may contain a 2′ modificationwith respect to a 2′ hydroxyl. In some embodiments the 2′ modificationmay be independently selected from O-alkyl (which may be substituted),halo, and deoxy (H).

The oligonucleotide may also contain one or more phosphorothioatelinkages. For example, the oligonucleotide may be fullyphosphorothioate-linked or may contain about half or ¾ phosphorotioatelinkages.

Exemplary oligonucleotide inhibitors are shown in Table 1.

In another aspect, the invention provides pharmaceutical compositionsand formulations comprising the oligonucleotides of the invention, whichmay involve incorporation of the oligonucleotide within a variety ofmacromolecular assemblies, micelle, or liposome compositions forcellular delivery. In certain embodiments, the oligonucleotides areformulated for conventional intravenous, subcutaneous, or intramusculardosing. Such formulations may be, conventional aqueous preparations,such as formulation in saline. In certain embodiments, the compositionsare suitable or formulated for intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection, or by direct injection intotarget tissue (e.g., cardiac tissue).

In still other aspects, the invention provides a method for deliveringoligonucleotides and the pharmaceutical compositions to mammalian cellseither in vitro or ex vivo, e.g., for treating, ameliorating, orpreventing the progression of a condition in a mammalian patient. Themethod may comprise administering the oligonucleotide or compositioncomprising the same to a mammalian patient or population of targetcells. The patient may have a condition associated with, mediated by, orresulting from, miR-208 family expression. Such conditions include, forexample, cardiac hypertrophy, myocardial infarction, heart failure(e.g., congestive heart failure), vascular damage, restenosis, orpathologic cardiac fibrosis. Thus, the invention provides a use of themodified oligonucleotides and compositions of the invention for treatingsuch conditions, and for the preparation of medicaments for suchtreatments.

Other aspects and embodiments of the invention will be apparent from thefollowing detailed description of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1. The psiCHECK™-2 construct (Promega) for quantifying inhibitoractivity in vitro using the dual luciferase assay.

FIG. 2. miR-208 inhibitor efficacy measured by Dual Luciferase assay formiR-208a. FIG. 2 shows the effect of adding LNAs: 10673 has 9 LNAs of 16nucleotides (9/16), 10674 has 11/16, 10677 has 13/16, and 10101 and10591 have 9/16. “208 alone” refers to the luciferase construct alonehaving the miR-208a recognition site cloned 3′ to the renilla luciferasegene. “208+mimic” includes cotransfection of mir-208a.

FIG. 3. miR-208 inhibitor efficacy measured by Dual Luciferase assay formiR-208a. M-10591 is a non-targeting control.

FIG. 4. miR-208 inhibitor efficacy measured by Dual Luciferase assay formiR-208b.

FIG. 5. miR-208 inhibitor efficacy measured by Dual Luciferase assay formiR-499.

FIG. 6. miR-208a and miR-208b expression levels in the heart after invivo dosing of miRNA inhibitor designs in normal mice. Left bars aremiR-208a expression levels and right bars are miR-208b expressionlevels.

FIG. 7. Survival of Dahl salt-sensitive rats after in vivo dosing ofmiRNA inhibitor designs at 25 mg/kg subcutaneously every two weeks.

FIG. 8. Percent body weight changes for Dahl salt-sensitive rats dosedwith inhibitor designs as in FIG. 7.

FIG. 9. Chart showing placement of Locked Nucleic Acids in the top 10miR-208 inhibitor designs (SEQ ID NOS:94-99).

FIG. 10. miR-208a abundance in heart determined by Real-time PCR afterin vivo dosing of miR-208a inhibitor designs in normal mice.

FIG. 11. miR-208b abundance in heart determined by Real-time PCR afterin vivo dosing of miR-208a inhibitor designs in normal mice.

FIG. 12. Systemic delivery of antimiR-208a (M-10101) induces potent andsustained silencing of miR-208 in the heart, FIG. 12 shows Real-time PCRanalysis on murine hearts one week after intravenous (i.v.) delivery ofincreasing, doses of antimiR-208a, and demonstrates a dose-dependentreduction in miR-208a levels. n=4 for each dose.

FIG. 13. Real-time PCR analysis on cardiac tissue collected at theindicated time-points. FIG. 13 shows that i.v., intraperitoneal (i.p) orsubcutaneous (s.c.) delivery of 25 mg/kg of antimiR-208a (M-10101)induces potent silencing of miR-208a. n=4 for each group.

FIG. 14. miR-208a silencing reduces miR-499 and Myh7. FIG. 14A showsReal-time PCR analysis demonstrating that antimiR-208a (M-10101)potently reduces cardiac levels of miR-208a up to 6 weeks afterinjection, which leads to a time-responsive reduction in miR-499. Dosingwith both an antimiR against miR-208a and -499 induces an immediatereduction in cardiac levels of both miR-208a and miR-499. FIG. 14B shows(by real-time PCR) that Myh7 is reduced 4 weeks after miR-208ainhibition, while inhibition of miR-208a and miR-499 reduces Myh7 after2 weeks. FIG. 14C is a Western blot analysis for Myh7 showing reducedMyh7 expression at the indicated time-points following antimiR-208a orantimiR-208a/-499 treatment. Gapdh serves as a loading control. ForFIGS. 14A and B, error bars depict SEM. n=4 for each time-point anddose.

FIG. 15. Tissue distribution analysis indicates that significant amountsof antimiR-208a (M-10101) are detectable in plasma, heart, liver ofkidney up to 6 weeks after injection. Error bars depict SEM. n=4 foreach time-point and dose.

FIG. 16. Therapeutic silencing of miR208a is beneficial during heartfailure. FIG. 16A shows Kaplan-Meier survival curves in the Dahlhypertensive rat model, and shows a pronounced decrease in survival inresponse to a HS diet for both HS/Saline and HS/Control groups, which issignificantly improved in response to antimiR-208a (M-10101) treatment.FIG. 16B shows body weight analysis, and indicates that Dahlhypertensive rats on 8% HS diet exhibit reduced weight gain compared toanimals on LS diet, while HS/antimiR-208a treated rats show asignificantly better maintenance in weight gain. For (a) and (b), n=6for LS/Saline; n=15 for HS/Saline and HS/Control; and n=14 forHS/antimiR-208a. The “n” on the graph represents total survivorsremaining at week 8 post-diet.

FIG. 17. Body weight analysis of Dahl rats on the 4% HS diet (FIG. 17A),showing significant reductions in weight gain compared to LS dietcontrols, while both 5 and 25 mg/kg injections every 2 weeks issufficient to maintain weight gain comparable to animals on a normaldiet. Error bars depict SEM, * p<0.05 vs. HS saline, # p<0.05 vs. LSsaline. FIG. 17B shows echocardiography measurements, indicating thatthe increase in IVRT and decrease in MV E/A in response to 4% HS dietare significantly improved in response to antimiR-208a treatment 8 weeksafter the onset of the diet. IVRT, isovolumic relaxation time; MV E/A,mitral valve early to active filling velocity ratio. n=10 for allgroups.

FIG. 18. Representative images of H&E and picrosirius red stained leftventricular histological sections indicate an increase in cardiomyocytehypertrophy and perivascular fibrosis in response to the 4% HS diet for8 weeks, while both parameters are reduced in response to antimiR-208a(M-10101) treatment (FIG. 18A). FIG. 18B is a bar-graph representationof histological quantification showing significantly less hypertrophyand fibrosis in the presence of antimiR-208a. Error bars depict SEM, *p<0.05 vs. HS saline, # p<0.05 vs. LS saline.

FIG. 19. antimiR-208a (M-10101) treatment reduces miR-499 and Myh7 inDahl salt-sensitive rats. All analyses were performed 8 weeks followingthe onset of 4% HS diet and 7 weeks after the onset of antimiRtreatment. n=10 for all groups in (a) and (c). FIG. 19A shows real-timePCR analysis indicating a dose-dependent reduction of miR-208a in bothleft ventricle (LV) and right ventricle (RV), which corresponds to adose-dependent decrease in miR-499. While miR-208b is increased inresponse to the HS diet, antimiR-208a significantly blunts thisresponse. Administration of a control chemistry (directed against a C.elegans miR) has no effect on the expression of either miR-208a, miR-499or miR-208b. Error bars depict SEM, * p<0.05 vs. HS saline, # p<0.05 vs.LS saline. FIG. 19B shows that regulation of miR-499 and miR-208b inresponse to antimiR-208a treatment can be confirmed by Northern blotanalysis. U6 serves as a loading control.

FIG. 20. Real-time PCR analysis showing that HS diet reduces Myh6, whileit increases Myh7 (FIG. 20A). AnitmiR-208a (M-10101) treatmentdose-dependently increases Myh6 expression while it reduces Myh7bexpression. The HS diet-induced increase in Myh7 is dose-dependentlyreduced by antimiR-208a. Error bars depict SEM, * p<0.05 vs. HS saline,# p<0.05 vs. LS saline. FIG. 20B shows a Western blot analysis for Myh7from ventricular tissue confirms the dose-dependent reduction inresponse to antimiR-208a treatment. Gapdh is used as a loading control.

FIG. 21. miR-499 in plasma serves a biomarker for antimiR-208a efficacy.FIG. 21 shows real-time PCR analysis on plasma samples, indicating anincrease in miR-499 in response to HS diet, while antimiR-208asignificantly lowers the detection of miR-499 in plasma 8 weeksfollowing the onset of 4% HS diet and 7 weeks after the onset of antimiRtreatment. Further miRNA analysis additionally indicates a decrease inplasma detectable miR-423-5p in response to antimiR-208a.

FIG. 22. Tissue and plasma distribution in African Green Monkeys (−3kg). Antimirs 10101 (antimiR-208a) and 10707 (antimiR-208b) wereadministered three times at a dose of 25 mg/kg by the saphenous vein,and drug plasma clearance determined (right panel). Tissue was collectedafter four weeks and assayed for inhibitor (dark bars, M-10101; lightbars, M-10707).

FIG. 23. Specific miRNA target inhibition in African Green Monkeys. Leftpanel shows changes in miR-208a expression in left ventricle (left toright: untreated, M-10101, M-10707, M-10591). Right panel shows changesin miR-208b in left ventricle (left to right: untreated, M-10101,M-10707, M-10591). As shown, with only two nucleotide differencesbetween M-10101 and M-10707, the antimiRs are specific for their targetmiR (miR-208a and miR-208b, respectively).

FIG. 24. Mir-499 levels after treatment. Levels are shown for leftventricle (LV), right ventricle (RV), and septum. Bars are, from left toright, untreated, M-10101, M-10707, and M-10591.

FIG. 25. antimiR-208a compounds with different chemistry patterns showmiR-208a knockdown in the left ventricle when administered to rats at 25mg/kg subcutaneously. The compounds show different levels of targetde-repression.

FIGS. 26 to 28. Target de-repression with antimiR-208a compounds as inFIG. 25.

FIG. 29. Antimir-208a treatment increases miR-19b plasma levels inunstressed rodents (SD rats).

FIG. 30. Studies with salt-sensitive rats show that the degree of targetde-repression depends on degree of stress. Dynlt1 shows more robustde-repression with 6% salt stress.

FIG. 31. Degree of target de-repression (Vcpip1) at different degrees ofstress (4% and 6% salt diets) in salt sensitive rat model.

FIG. 32. Degree of target de-repression (Tmbim6) at different degrees ofstress (4% and 6% salt diets) in salt sensitive rat model.

FIG. 33. Degree of miR inhibition in different regions of the heart,showing that more stressed regions show greater effect.

FIG. 34. Degree of myosin expression in different regions of the heartupon antimiR-208a treatment, showing that more stressed regions showgreater effect.

FIG. 35. Degree of expression of certain cardiac stress markers indifferent regions of the heart upon antimiR-208a treatment.

FIG. 36. Degree of target expression in different regions of the heartupon antimiR-208a treatment, showing that more stressed regions showgreater effect.

FIG. 37. Degree of target expression in different regions of the heartupon antimiR-208a treatment, showing that more stressed regions showgreater effect.

FIG. 38. Degree of Dynlt1 de-repression in different regions of theheart upon atimiR-208a treatment, showing that more stressed regionsshow greater effect.

FIG. 39. Degree of target de-repression in different regions of theheart upon atimiR-208a treatment, showing that more stressed regionsshow greater effect.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides chemically modified oligonucleotides capable ofinhibiting the expression (e.g., abundance) of miR-208 family miRNAs,including miR-208a, miR-208b, and/or miR-499. The invention provides insome embodiments, oligonucleotides capable of inhibiting, in a specificfashion, the expression or abundance of each of miR-208a, miR-208b, andmiR-499. The invention further provides pharmaceutical compositionscomprising the oligonucleotides, and methods of treating patients havingconditions or disorders relating to or involving a miR-208 family miRNA,such as a various cardiovascular conditions. In various embodiments, theoligonucleotides provide advantages in one or more of potency,efficiency of delivery, target specificity, toxicity, and/or stability.

Chemically-Modified miR-208a Antisense Oligonucleotides

In one aspect, the invention provides an oligonucleotide capable ofreducing the expression or abundance of miR-208 family miRNAs. Theactivity of the oligonucleotides may be determined in vitro and/or invivo. For example, when inhibition of miR-208a, miR-208b, or miR-499activity is determined in vitro, the activity may be determined using adual luciferase assay as described herein. The oligonucleotidesignificantly inhibits such activity, as determined in the dualluciferase activity, at a concentration of about 50 nM or less, or inother embodiments, 40 nM or less, 20 nM or less, or 10 nM or less. Forexample, the oligonucleotide may have an IC50 for inhibition ofmiR-208a, miR-208b, and/or miR-499 activity of about 50 nM or less, 40nM or less. 30 nM or less, or 20 nM or less, as determined in the dualluciferase assay.

The dual luciferase assay, as exemplified by the commercially availableproduct PsiCHECK™ (Promega), involves placement of the miR recognitionsite in the 3′ UTR of a gene for a detectable protein (e.g., renillaluciferase). The construct is co-expressed with the target miRNA, suchthat inhibitor activity can be determined by change in signal. A secondgene encoding a detectable protein (e.g., firefly luciferase) can beincluded on the same plasmid, and the ratio of signals determined as anindication of antimiR activity.

Alternatively, or in addition, the activity of the oligonucleotide maybe determined in a suitable mouse or rat model, such as those describedherein, where inhibition (e.g., by at least 50%) of a miR-208 familymiRNA is observed at an oligonucleotide dose of 50 mg/kg or less, 25mg/kg or less, such as 10 mg/kg or less or 5 mg/kg or less. In someembodiments, the activity of the oligonucleotides is determined in ananimal model described in WO 2008/016924, which descriptions are herebyincorporated by reference. For example, the oligonucleotide may exhibitat least 50% target miRNA inhibition or target de-repression at a doseof 50 mg/kg or less, 25 mg/kg or less, such as 10 mg/kg or less or 5mg/kg or less. In such embodiments, the oligonucleotide may be dosedintravenously or subcutaneously to mice, and the oligonucleotide may beformulated in saline.

In these or other embodiments, the oligonucleotides of the invention arestable after administration, being detectable in the circulation and/ortarget organ for at least three weeks, at least four weeks, at leastfive weeks, or at least six weeks, or more, following administration.Thus, the oligonucleotides of the invention have the potential toprovide for less frequent administration, lower doses, and/or longerduration of therapeutic effect.

The nucleotide sequence of the oligonucleotide: is substantiallycomplementary to a nucleotide sequence of human miR-208a and/ormiR-208b, and contains a mix of locked and non-locked nucleotides. Forexample, the oligonucleotide may contain at least five or at least sevenor at least nine locked nucleotides, and at least one non-lockednucleotide. Generally, the length of the oligonucleotide and number andposition of locked nucleotides is such that the oligonucleotide reducesmiR-208a, miR-208b, and/or miR-499 activity at an oligonucleotideconcentration of about 50 nM or less in the in vitro luciferase assay,or at a dose of about 50 mg/kg or less, or about 25 mg/kg or less in asuitable mouse or rat model, each as described. A substantiallycomplementary oligonucleotide may have from 1 to 4 mismatches (e.g., 1or 2 mismatches) with respect to its target sequence of miR-208a ormiR-208b.

miR-208a, including its structure and processing, and its potential fortreating cardiac hypertrophy, heart failure, or myocardial infarction(among other conditions), are described in WO 2008/016924, which ishereby incorporated by reference in its entirety. The pre-miRNA sequencefor human miR-208a, which may be used for designing inhibitory miRNAs inaccordance with the invention, is (the underlined sequence is the matureform):

(SEQ ID NO: 5) 5′- ACGGGCGAGC UUUUGGCCCG GGUUAUACCU GAUGCUCACGUAUAAGACGA GCAAAAAGCU UGUUGGUCAG A -3′

The structure and processing of miR-208b and miR-499 are also describedin WO 2009/018492, which is hereby incorporated by reference. MaturemiR-208b has the nucleotide sequence 5′-AUGAAGACGAACAAAAGGUUUGU-3′ (SEQID NO:6), and mature miR-499 has the nucleotide sequence5′-UUAAGACUUGCAGUGAUGUUU-3′ (SEQ ID NO:7). These sequences may be usedto design complementary inhibitors in accordance with the invention.

The oligonucleotide contains one or more locked nucleic acid (LNAs)residues, or “locked nucleotides”, LNAs are described, for example, inU.S. Pat. No. 6,268,490, U.S. Pat. No. 6,316,198, U.S. Pat. No.6,403,566, U.S. Pat. No. 6,770,748, U.S. Pat. No. 6,998,484, U.S. Pat.No. 6,670,461, and U.S. Pat. No. 7,034,133, all of which are herebyincorporated by reference in their entireties. LNAs are modifiednucleotides or ribonucleotides that contain an extra bridge between the2′ and 4′ carbons of the ribose sugar moiety resulting in a “locked”conformation, and/or bicyclic structure. In one embodiment, theoligonucleotide contains one or more LNAs having the structure shown bystructure A below. Alternatively or in addition, the oligonucleotide maycontain one or more LNAs having the structure shown by structure Bbelow. Alternatively or in addition, the oligonucleotide contains one ormore LNAs having the structure shown by structure C below.

Other suitable locked nucleotides that can be incorporated in theoligonucleotides of the invention include those described in U.S. Pat.No. 6,403,566 and U.S. Pat. No. 6,833,361, both of which are herebyincorporated by reference in their entireties.

In exemplary embodiments, the locked nucleotides have a 2′ to 4′methylene bridge, as shown in structure A, for example.

The oligonucleotide may comprise, consist essentially of, or consist of,a full length or truncated miR-208a or miR-208b antisense sequence. Asused herein, the term “full length” in reference to a miRNA sequencerefers to the length of the mature miRNA antisense counterpart. Thus,the inhibitors described herein may be truncated or full-length,antisense, mature miRNA sequences, or may comprise these sequences incombination with other polynucleotide sequences. In certain embodiments,the chemical modification motif described herein renders full lengthantisense miRNA (mature) sequences unnecessary. In these embodiments,the oligonucleotide is from 8 to 20 nucleotides in length, or is from 10to 18 nucleotides in length, or is from 11 to 16 nucleotides in length.The oligonucleotide in some embodiments is about 12, about 13, about 14,about 15, about 16, about 17, or about 18 nucleotides in length. Thetruncated oligonucleotide may have a sequence that targets, by antisenseinhibition, a miR-208a sequence within 5′-UAAGACGAGCAAAAAG-3′ (SEQ IDNO:8) or a miR-208b sequence within UAAGACGAACAAAAAG-3′ (SEQ ID NO:9).

The oligonucleotide generally has a nucleotide sequence designed totarget mature miR-208a, miR-208b, and/or miR-499. The oligonucleotidemay, in these or other embodiments, also or alternatively be designed totarget the pre- or pri-miRNA forms. In certain embodiments, theoligonucleotide may be designed to have a sequence containing from 1 to5 (e.g., 1, 2, 3, or 4) mismatches relative to the fully complementary(mature) miR-208 sequence. In certain embodiments, such antisensesequences may be incorporated into shRNAs or other RNA structurescontaining stem and loop portions, for example.

In certain embodiments, the oligonucleotide comprises a nucleotidesequence that is completely complementary to a nucleotide sequence ofmiR-208a or miR-208b. For example, the oligonucleotide may comprise thenucleotide sequence of 5′-TGCTCGTCTTA-3′ (SEQ ID NO:1) or may comprisethe nucleotide sequence of 5′-TGTTCGTCTTA 3′ (SEQ ID NO:2). Inparticular embodiments, the oligonucleotide comprises, consistsessentially of, or consists of the nucleotide sequence5′-CTTTTTGCTCGTCTTA-3′ (SEQ ID NO:3) or 5-CCTTTTGTTCGTCTTA (SEQ IDNO:4). In this context, “consists essentially of” includes the optionaladdition of nucleotides (e.g., one or two) on either or both of the 5′and 3′ ends, so long as the additional nucleotide(s) do notsubstantially affect (as defined by an increase in IC50 of no more than20%) the oligonucleotide's inhibition of the target miRNA activity inthe dual luciferase assay or mouse model.

The oligonucleotide generally contains at least 3, at least 5, at least7, or at least 9 locked nucleotides, but in various embodiments is notfully comprised of locked nucleotides. Generally, the number andposition of locked nucleotides is such that the oligonucleotide reducesmiR-208a, miR-208b, and/or miR-499 activity as determined in vitro or invivo as described. In certain embodiments, the oligonucleotide does notcontain a stretch of nucleotides with more than four, or more thanthree, contiguous non-locked nucleotides. In certain embodiments, theoligonucleotide does not contain a stretch of nucleotides with more thantwo contiguous non-locked nucleotides. For example, the oligonucleotidemay have just one occurrence of contiguous non-locked nucleotides. Inthese or other embodiments, the region complementary to the miR-208a,miR-208b, and/or miR-499 seed region comprises at least three or atleast four locked nucleotides. These embodiments may, for example,employ a nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4.

Thus, in various embodiments, the oligonucleotide contains at least ninelocked nucleotides, or at least eleven locked nucleotides. Theoligonucleotide may contain at least three or at least 5 non-lockednucleotides. For example, the oligonucleotide may contain nine lockednucleotides and seven non-locked nucleotides, or may contain elevenlocked nucleotides and five non-locked nucleotides.

The pattern of locked nucleotides may be such that at least positions 1,6, 10, 13, and 15 are locked nucleotides. In certain embodiments,positions 1, 5, 6, 8, 10, 11, 13, 15, and 16 are locked nucleotides, andthe remaining positions are non-locked nucleotides. In otherembodiments, positions 1, 3, 4, 5, 6, 8, 10, 13, 15, and 16 are lockednucleotides, with the remaining positions being non-locked nucleotides.In some embodiments, positions 1, 4, 5, 7, 9, 10, 12, 14, and 16 arelocked nucleotides, and remaining positions are non-locked nucleotides.In exemplary embodiments, such patterns find use with an oligonucleotidehaving the sequence of SEQ ID NO:3 or SEQ ID NO:4.

For non-locked nucleotides, the nucleotide may contain a 2′ modificationwith respect to a 2′ hydroxyl. For example, the 2′ modification may be2′ deoxy. Incorporation of 2′-modified nucleotides in antisenseoligonucleotides may increase both resistance of the oligonucleotides tonucleases and their thermal stability with complementary RNA. Variousmodifications at the 2′ positions may be independently selected fromthose that provide increased nuclease sensitivity, without compromisingmolecular interactions with the RNA target or cellular machinery. Suchmodifications may be selected on the basis of their increased potency invitro or in vivo. Exemplary methods for determining increased potency(e.g., IC50) for miRNA inhibition are described herein, including thedual luciferase assay and in vivo miRNA expression or targetde-repression.

In some embodiments the 2′ modification may be independently selectedfrom O-alkyl (which may be substituted), halo, and deoxy (H).Substantially all, or all, nucleotide 2′ positions of the non-lockednucleotides may be modified in certain embodiments, e.g., asindependently selected from O-alkyl (e.g., O-methyl), halo (e.g.,fluoro), deoxy (H), and amino. For example, the 2′ modifications mayeach be independently selected from O-methyl and fluoro. In exemplaryembodiments, purine nucleotides each have a 2′ OMe and pyrimidinenucleotides each have a 2′-F. In certain embodiments, from one to aboutfive 2′ positions, or from about one to about three 2′ positions areleft unmodified (e.g., as 2′ hydroxyls).

2′ modifications in accordance with the invention also include smallhydrocarbon substituents. The hydrocarbon substituents include alkyl,alkenyl, alkynyl, and alkoxyalkyl, where the alkyl (including the alkylportion of alkoxy), alkenyl and alkynyl may be substituted orunsubstituted. The alkyl, alkenyl, and alkynyl may be C1 to C10 alkyl,alkenyl or alkynyl, such as C1, C2, or C3. The hydrocarbon substituentsmay include one or two or three non-carbon atoms, which may beindependently selected from N, O, and/or S. The 2′ modifications mayfurther include the alkyl, alkenyl, and alkynyl as O-alkyl, 0-alkenyl,and O-alkynyl.

Exemplary 2′ modifications in accordance with the invention include2′-O-alkyl (C1-3 alkyl, such as 2′OMe or 2′OEt), 2′-O-methoxyethyl(2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) substitutions.

In certain embodiments, the oligonucleotide contains at least one2′-halo modification (e.g., in place of a 2′ hydroxyl), such as2′-fluoro, 2′-chloro, 2′-bromo, and 2′-iodo. In some embodiments, the 2′halo modification is fluoro. The oligonucleotide may contain from 1 toabout 5 2′-halo modifications (e.g., fluoro), or from 1 to about 32′-halo modifications (e.g., fluoro). In some embodiments, theoligonucleotide contains all 2′-fluoro nucleotides at non-lockedpositions, or 2′-fluoro on all non-locked pyrimidine nucleotides. Incertain embodiments, the 2′-fluoro groups are independently di-, tri-,or un-methylated.

The oligonucleotide may have one or more 2′-deoxy modifications (e.g., Hfor 2′ hydroxyl), and in some embodiments, contains from 2 to about 102′-deoxy modifications at non-locked positions, or contains 2′ deoxy atall non-locked positions.

In exemplary embodiments, the oligonucleotide contains 2′ positionsmodified as 2′OMe in non-locked positions. Alternatively, non-lockedpurine nucleotides are modified at the 2′ position as 2′OMe, withnon-locked pyrimidine nucleotides modified at the 2′ position as2′-fluoro.

In certain embodiments, the oligonucleotide further comprises at leastone terminal modification or “cap”. The cap may be a 5′ and/or a 3′-capstructure. The terms “cap” or “end-cap” include chemical modificationsat either terminus of the oligonucleotide (with respect to terminalribonucleotides), and including modifications at the linkage between thelast two nucleotides on the 5′ end and the last two nucleotides on the3′ end. The cap structure as described herein may increase resistance ofthe oligonucleotide to exonucleases without compromising molecularinteractions with the RNA target or cellular machinery. Suchmodifications may be selected on the basis of their increased potency invitro or in vivo. The cap can be present at the 5′-terminus (5′-cap) orat the 3′-terminus (3′-cap) or can be present on both ends. In certainembodiments, the 5′- and/or 3′-cap is independently selected fromphosphorothioate monophosphate, abasic residue (moiety),phosphorothioate linkage, 4′-thio nucleotide, carbocyclic nucleotide,phosphorodithioate linkage, inverted nucleotide or inverted abasicmoiety (2′-3′ ear 3′-3′), phosphorodithioate monophosphate, andmethylphosphonate moiety. The phosphorothioate or phosphorodithioatelinkage(s), when part of a cap structure, are generally positionedbetween the two terminal nucleotides on the 5′ end and the two terminalnucleotides on the 3′ end.

In certain embodiments, the oligonucleotide has at least one terminalphosphorothioate monophosphate. The phosphorothioate monophosphate maysupport a higher potency by inhibiting the action of exonucleases. Thephosphorothioate monophosphate may be at the 5′ and/or 3′ end of theoligonucleotide. A phosphorothioate monophosphate is defined by thefollowing structures, where B is base, and R is a 2′ modification asdescribed above:

Where the cap structure can support the chemistry of a lockednucleotide, the cap structure may incorporate a locked nucleotide asdescribed herein.

Phosphorothioate linkage's may be present in some embodiments, such asbetween the last two nucleotides on the 5′ and the 3′ end (e.g., as partof a cap structure), or as alternating with phosphodiester bonds. Inthese or other embodiments, the oligonucleotide may contain at least oneterminal abasic residue at either or both the 5′ and 3′ ends. An abasicmoiety does not contain a commonly recognized purine or pyrimidinenucleotide base, such as adenosine, guanine, cytosine, uracil orthymine. Thus, such abasic moieties lack a nucleotide base or have othernon-nucleotide base chemical groups at the 1′ position. For example, theabasic nucleotide may be a reverse abasic nucleotide, e.g., where areverse abasic phosphoramidite is coupled via a 5′ amidite (instead of3′ amidite) resulting in a 5′-5′ phosphate bond. The structure of areverse abasic nucleoside for the 5′ and the 3′ end of a polynucleotideis shown below.

The oligonucleotide may contain one or more phosphorothioate linkages.Phosphorothioate linkages have been used to render oligonucleotides moreresistant to nuclease cleavage. For example, the polynucleotide may bepartially phosphorothioate-linked, for example, phosphorothioatelinkages may alternate with phophodiester linkages. In certainembodiments, however, the oligonucleotide is fullyphosphorothioate-linked. In other embodiments, the oligonucleotide hasfrom one to five or one to three phosphate linkages.

In some embodiments, the nucleotide has one or more carboxamido-modifiedbases as described M PCT/US11/59588, which is hereby incorporated byreference, including with respect to all exemplary pyrimidinecarboxamido modifications disclosed therein with heterocyclicsubstituents.

In exemplary embodiments, the oligonucleotide has the structure of aCompound listed in Table 1, below.

TABLE 1 Exemplary Oligonucleotides Cmpd# (M) Alias Sequence Length 10101208a_DNA_LNA_16_PS 5′ lCs; dTs; dTs; dTs; lTs; lTs; dGs; lCs; dTs; lCs;lGs; 3′ 16 dTs; lCs; dTs; lTs; lA (SEQ ID NO: 10) 10570 208fam optdes15′ lTs; dGs; lCs; lTs; lCs; dGs; lTs; lCs; dTs; lTs; lA 3′ 11 (SEQ IDNO: 11) 10571 208fam optdes2 5′ lTs; dGs; lCs; lTs; lCs; dGs; dTs; lCs;dTs; lTs; lA 3′ 11 (SEQ ID NO: 12) 10572 208fam optdes3 5′ lTs; dGs;lCs; dAs; lCs; dGs; lTs; dCs; lTs; lTs; lA 3′ 11 (SEQ ID NO: 13) 10573208fam optdes4 5′ lTs; lGs; dCs; dAs; lCs; lGs; dTs; lCs; dTs; lTs; lA3′ 11 (SEQ ID NO: 14) 10673 208a LNA 5′ lCs; dTs; lTs; lTs; lTs; lTs;dGs; lCs; dTs; lCs; dGs; 3′ 16 C T DNA 16 1 dTs; lCs; dTs; lTs; dA (SEQID NO: 15) 10674 208a_LNA 5′ lCs; dTs; dTs; lTs; lTs; lTs; dGs; lCs;lTs; lCs; dGs; 3′ 16 C T DNA 16 2 lTs; lCs; lTs; lTs; dA (SEQ ID NO: 16)10677 208a_LNA 5′ lCs; lTs; lTs; lTs; lTs; lTs; dGs; lCs; lTs; lCs; dGs;3′ 16 C T DNA 16 3 lTs; lCs; lTs; lTs; dA (SEQ ID NO: 17) 10679 208 LNAopt 1 5′ lCs; dTs; lTs; dTs; lTs; lTs; dGs; lCs; dTs; lCs; dGs; 3′ 16lTs; dCs; lTs; lTs; dA (SEQ ID NO: 18) 10680 208 LNA opt 2 5′ lCs; dTs;lTs; lTs; lTs; lTs; dGs; lCs; dTs; lCs; dGs; 3′ 16 dTs; lCs; dTs; dTs;lA (SEQ ID NO: 19) 10681 208 LNA opt 3 5′ lCs; dTs; lTs; lTs; dTs; lTs;dGs; lCs; lTs; lCs; dGs; 3′ 16 dTs; lCs; dTs; lTs; dA (SEQ ID NO: 20)10682 208 LNA opt 4 5′ lCs; dTs; lTs; dTs; lTs; dTs; lGs; dCs; lTs; dCs;lGs; 3′ 16 dTs; lCs; dTs; lTs; lA (SEQ ID NO: 21) 10683 208 LNA opt 5 5′lCs; dTs; dTs; lTs; lTs; dTs; lGs; dCs; lTs; lCs; dGs; 3′ 16 lTs; dCs;lTs; dTs; lA (SEQ ID NO: 22) 10707 208b_DNA_LNA_16_PS 5′ lCs; dCs; dTs;dTs; lTs; lTs; dGs; lTs; dTs; lCs; lGs; 3′ 16 dTs; lCs; dTs; lTs; lA(SEQ ID NO: 23) 10718 208a like 15 1 5′ lTs; lTs; lTs; lTs; lTs; dGs;lCs; dTs; lCs; dGs; dTs; 3′ 15 lCs; dTs; lTs; dA (SEQ ID NO: 24) 10719208a like 15 2 5′ lTs; lTs; dTs; lTs; lTs; dGs; lCs; dTs; lCs; dGs; dTs;3′ 15 lCs; dTs; lTs; dA (SEQ ID NO: 25) 10720 208a like 15 3 5′ lTs;lTs; lTs; lTs; lTs; dGs; dCs; dTs; lCs; dGs; lTs; 3′ 15 lCs; dTs; lTs;dA (SEQ ID NO: 26) 10721 208a like 15 4 5′ lTs; dTs; lTs; lTs; lTs; dGs;lCs; dTs; lCs; dGs; dTs; 3′ 15 lCs; lTs; lTs; dA (SEQ ID NO: 27) 10722208a like 15 5 5′ lTs; lTs; lTs; lTs; lTs; dGs; lCs; dTs; lCs; dGs; dTs;3′ 15 lCs; lTs; lTs; lA (SEQ ID NO: 28) 10723 208a like 15 6 5′ lTs;dTs; lTs; lTs; lTs; dGs; lCs; dTs; lCs; dGs; dTs; 3′ 15 lCs; lTs; lTs;lA (SEQ ID NO: 29) 10724 208b like 15 1 5′ lCs; lTs; lTs; lTs; lTs; dGs;lCs; dTs; lCs; dGs; dTs; 3′ 15 lCs; dTs; lTs; dA (SEQ ID NO: 30) 10725208b like 15 2 5′ lCs; lTs; dTs; lTs; lTs; dGs; lCs; dTs; lCs; dGs; dTs;3′ 15 lCs; dTs; lTs; dA (SEQ ID NO: 31) 10726 208b like 15 3 5′ lCs;dTs; lTs; lTs; lTs; dGs; lCs; dTs; lCs; dGs; dTs; 3′ 15 lCs; lTs; lTs;dA (SEQ ID NO: 32) 10727 208b like 15 4 5′ lCs; lTs; lTs; lTs; lTs; dGs;lCs; dTs; lCs; dGs; dTs; 3′ 15 lCs; dTs; lTs; lA (SEQ ID NO: 33) 10728208b like 15 5 5′ lCs; dTs; lTs; lTs; lTs; dGs; lCs; dTs; lCs; dGs; dTs;3′ 15 lCs; dTs; lTs; lA (SEQ ID NO: 34) 10729 208b like 15 6 5′ lCs;lTs; lTs; lTs; lTs; dGs; dCs; dTs; lCs; dGs; lTs; 3′ 15 lCs; dTs; lTs;dA (SEQ ID NO: 35) 10730 208b 15 1 5′ lCs; lTs; lTs; lTs; lTs; dGs; lTs;dTs; lCs; dGs; dTs; 3′ 15 lCs; dTs; lTs; dA (SEQ ID NO: 36) 10731 208b15 2 5′ lCs; lTs; lTs; lTs; lTs; dGs; lTs; dTs; lCs; dGs; dTs; 3′ 15lCs; dTs; lTs; lA (SEQ ID NO: 37) 10732 208b 15 3 5′ lCs; lTs; lTs; lTs;lTs; dGs; lTs; dTs; lCs; dGs; lTs; 3′ 15 lCs; dTs; lTs; dA (SEQ ID NO:38) 10733 208a like 15 7 5′ lTs; dTs; lTs; dTs; lTs; dGs; dCs; dTs; lCs;lGs; lTs; 3′ 15 lCs; lTs; lTs; lA (SEQ ID NO: 39) 10734 208b like 15 75′ lCs; dTs; lTs; dTs; lTs; dGs; dCs; dTs; lCs; lGs; lTs; 3′ 15 lCs;lTs; lTs; lA (SEQ ID NO: 40) 10735 208b 15 4 5′ lCs; dTs; lTs; dTs; lTs;dGs; dTs; dTs; lCs; lGs; lTs; 3′ 15 lCs; lTs; lTs; lA (SEQ ID NO: 41)10736 208a like 14 1 5′ lTs; lTs; lTs; lTs; dGs; lCs; dTs; lCs; dGs;dTs; lCs; 3′ 14 dTs; lTs; dA (SEQ ID NO: 42) 10737 208a like 14 2 5′lTs; lTs; lTs; lTs; dGs; lCs; dTs; lCs; dGs; dTs; lCs; 3′ 14 dTs; lTs;lA (SEQ ID NO: 43) 10738 208a like 14 3 5′ lTs; lTs; lTs; lTs; dGs; dCs;dTs; lCs; dGs; dTs; lCs; 3′ 14 dTs; lTs; lA (SEQ ID NO: 44) 10739 208alike 14 4 5′ lTs; lTs; lTs; lTs; dGs; dCs; dTs; lCs; dGs; lTs; lCs; 3′14 dTs; lTs; lA (SEQ ID NO: 45) 10740 208a like 14 5 5′ lTs; lTs; lTs;lTs; dGs; dCs; lTs; lCs; dGs; lTs; dCs; 3′ 14 lTs; lTs; dA (SEQ ID NO:46) 10741 208a like 14 6 5′ lTs; dTs; lTs; dTs; dGs; lCs; dTs; lCs; lGs;lTs; lCs; 3′ 14 lTs; lTs; lA (SEQ ID NO: 47) 10742 208b 14 1 5′ lTs;lTs; lTs; lTs; dGs; lTs; dTs; lCs; dGs; dTs; lCs; 3′ 14 dTs; lTs; dA(SEQ ID NO: 48) 10743 208b 14 2 5′ lTs; lTs; lTs; lTs; dGs; lTs; dTs;lCs; dGs; dTs; lCs; 3′ 14 dTs; lTs; lA (SEQ ID NO: 49) 10744 208b 14 35′ lTs; lTs; lTs; lTs; dGs; dTs; dTs; lCs; dGs; dTs; lCs; 3′ 14 dTs;lTs; lA (SEQ ID NO: 50) 10745 208b 14 4 5′ lTs; lTs; lTs; lTs; dGs; dTs;dTs; lCs; dGs; lTs; lCs; 3′ 14 dTs; lTs; lA (SEQ ID NO: 51) 10746 208b14 5 5′ lTs; lTs; lTs; lTs; dGs; dTs; lTs; lCs; dGs; lTs; dCs; 3′ 14lTs; lTs; dA (SEQ ID NO: 52) 10747 208b 14 6 5′ lTs; dTs; lTs; dTs; dGs;lTs; dTs; lCs; lGs; lTs; lCs; 3′ 14 lTs; lTs; lA (SEQ ID NO: 53) 10748208a like 13 1 5′ lTs; lTs; lTs; dGs; lCs; dTs; lCs; dGs; dTs; lCs; dTs;3′ 13 lTs; dA (SEQ ID NO: 54) 10749 208a like 13 2 5′ lTs; lTs; lTs;dGs; lCs; dTs; lCs; dGs; dTs; lCs; dTs; 3′ 13 lTs; lA (SEQ ID NO: 55)10750 208a like 13 3 5′ lTs; lTs; lTs; dGs; lCs; dTs; lCs; lGs; lTs;lCs; lTs; 3′ 13 lTs; lA (SEQ ID NO: 56) 10751 208a like 13 4 5′ lTs;dTs; lTs; dGs; lCs; dTs; lCs; lGs; lTs; lCs; lTs; 3′ 13 lTs; lA (SEQ IDNO: 57) 10752 208b 13 1 5′ lTs; lTs; lTs; dGs; lTs; dTs; lCs; dGs; dTs;lCs; dTs; 3′ 13 lTs; dA (SEQ ID NO: 58) 10753 208b 13 2 5′ lTs; lTs;lTs; dGs; lTs; dTs; lCs; dGs; dTs; lCs; dTs; 3′ 13 lTs; lA (SEQ ID NO:59) 10754 208b 13 3 5′ lTs; lTs; lTs; dGs; lTs; dTs; lCs; lGs; lTs; lCs;lTs; 3′ 13 lTs; lA (SEQ ID NO: 60) 10755 208b 13 4 5′ lTs; dTs; lTs;dGs; lTs; dTs; lCs; lGs; lTs; lCs; lTs; 3′ 13 lTs; lA (SEQ ID NO: 61)10756 208a like 11 1 5′ lTs; dGs; lCs; dTs; lCs; dGs; dTs; lCs; dTs;lTs; dA 3′ 11 (SEQ ID NO: 62) 10757 208a like 11 2 5′ lTs; dGs; lCs;dTs; lCs; dGs; dTs; lCs; dTs; lTs; lA 3′ 11 (SEQ ID NO: 63) 10758 208b11 1 5′ lTs; dGs; lTs; dTs; lCs; dGs; dTs; lCs; dTs; lTs; dA 3′ 11 (SEQID NO: 64) 10759 208b 11 2 5′ lTs; dGs; lTs; dTs; lCs; dGs; dTs; lCs;dTs; lTs; lA 3′ 11 (SEQ ID NO: 65) 10760 208b 16 1 5′ lCs; dCs; lTs;lTs; lTs; lTs; dGs; lTs; dTs; lCs; dGs; 3′ 16 dTs; lCs; dTs; lTs; dA(SEQ ID NO: 66) 10761 208b 16 2 5′ lCs; dCs; lTs; dTs; lTs; lTs; dGs;lTs; dTs; lCs; dGs; 3′ 16 lTs; dCs; lTs; lTs; dA (SEQ ID NO: 67) 10762208b 16 3 5′ lCs; dCs; lTs; lTs; lTs; lTs; dGs; lTs; dTs; lCs; dGs; 3′16 dTs; lCs; dTs; dTs; lA (SEQ ID NO: 68) 10763 208b like 16 1 5′ lCs;dCs; lTs; lTs; lTs; lTs; dGs; lCs; dTs; lCs; dGs; 3′ 16 dTs; lCs; dTs;lTs; dA (SEQ ID NO: 69) 10764 208b like 16 2 5′ lCs; dCs; lTs; dTs; lTs;lTs; dGs; lCs; dTs; lCs; dGs; 3′ 16 lTs; dCs; lTs; lTs; dA (SEQ ID NO:70) 10765 208b like 16 3 5′ lCs; dCs; lTs; lTs; lTs; lTs; dGs; lCs; dTs;lCs; dGs; 3′ 16 dTs; lCs; dTs; dTs; lA (SEQ ID NO: 71) 10775 208b 15 55′ lTs; lTs; lTs; lTs; dGs; lTs; dTs; lCs; dGs; dTs; lCs; 3′ 15 dTs;lTs; dAs; lT (SEQ ID NO: 72) 10776 208b 15 6 5′ lTs; lTs; lTs; lTs; dGs;lTs; dTs; lCs; dGs; dTs; lCs; 3′ 15 dTs; lTs; lAs; lT (SEQ ID NO: 73)10777 208b 15 7 5′ lTs; lTs; lTs; lTs; dGs; lTs; dTs; lCs; dGs; lTs;lCs; 3′ 15 dTs; lTs; dAs; lT (SEQ ID NO: 74) 10778 208b 15 8 5′ lTs;lTs; dTs; lTs; dGs; dTs; dTs; lCs; lGs; lTs; lCs; 3′ 15 lTs; lTs; lAs;lT (SEQ ID NO: 75) 10779 208b 15 9 5′ lTs; lTs; lTs; lTs; dGs; lTs; dTs;lCs; dGs; dTs; lCs; 3′ 15 dTs; lTs; lAs; dT (SEQ ID NO: 76) 10780 208b15 10 5′ lTs; lTs; lTs; lTs; dGs; lTs; dTs; lCs; dGs; lTs; lCs; 3′ 15dTs; lTs; lAs; dT (SEQ ID NO: 77) 10781 208b 15 11 5′ lTs; lTs; dTs;lTs; dGs; lTs; dTs; lCs; lGs; lTs; lCs; 3′ 15 lTs; lTs; lAs; dT (SEQ IDNO: 78) 10782 208b 15 12 5′ lTs; lTs; lTs; lTs; dGs; dTs; dTs; lCs; dGs;dTs; lCs; 3′ 15 lTs; lTs; dAs; lT (SEQ ID NO: 79) 10783 208b_15_13 5′lTs; lTs; lTs; lTs; dGs; dTs; dTs; lCs; dGs; dTs; 3′ 15 lCs; lTs; lTs;lAs; dT (SEQ ID NO: 80) 10784 208b 14 7 5′ lTs; lTs; lTs; dGs; lTs; dTs;lCs; dGs; dTs; lCs; dTs; 3′ 14 lTs; dAs; lT (SEQ ID NO: 81) 10785 208b14 8 5′ lTs; lTs; lTs; dGs; lTs; dTs; lCs; dGs; dTs; lCs; dTs; 3′ 14lTs; lAs; lT (SEQ ID NO: 82) 10786 208b 14 9 5′ lTs; lTs; lTs; dGs; lTs;dTs; lCs; dGs; lTs; lCs; dTs; 3′ 14 lTs; dAs; lT (SEQ ID NO: 83) 10787208b 14 10 5′ lTs; dTs; lTs; dGs; dTs; dTs; lCs; lGs; lTs; lCs; lTs; 3′14 lTs; lAs; lT (SEQ ID NO: 84) 10788 208b 14 11 5′ lTs; lTs; lTs; dGs;lTs; dTs; lCs; dGs; dTs; lCs; dTs; 3′ 14 lTs; lAs; dT (SEQ ID NO: 85)10789 208b 14 12 5′ lTs; lTs; lTs; dGs; lTs; dTs; lCs; dGs; lTs; lCs;dTs; 3′ 14 lTs; lAs; dT (SEQ ID NO: 86) 10790 208b 14 13 5′ lTs; dTs;lTs; dGs; lTs; dTs; lCs; lGs; lTs; lCs; lTs; 3′ 14 lTs; lAs; dT (SEQ IDNO: 87) 10791 208b 14 14 5′ lTs; lTs; lTs; dGs; dTs; dTs; lCs; dGs; dTs;lCs; lTs; 3′ 14 lTs; dAs; lT (SEQ ID NO: 88) 10792 208b 14 15 5′ lTs;lTs; lTs; dGs; dTs; dTs; lCs; dGs; dTs; lCs; lTs; 3′ 14 lTs; lAs; dT(SEQ ID NO: 89) 10793 208b 16 4 5′ lCs; dTs; lTs; lTs; lTs; lGs; dTs;lTs; dCs; lGs; dTs; 3′ 16 dCs; lTs; dTs; lAs; dT (SEQ ID NO: 90) 111845′ lCs; dTs; lTs; lTs; dTs; dTs; lGs; lCs; dTs; lCs; dGs; 3′ 16 lTs;dCs; lTs; dTs; lAs (SEQ ID NO: 91)

TABLE 2 Description of Notations deoxy A dA deoxy G dG deoxy C dC deoxyT dT lna A lA lnaG lG lna C lC lna T lT deoxy A P = S dAs deoxy G P = SdGs deoxy C P = S dCs deoxy T P = S dTs lna A P = S lAs lnaG P = S lGslna C P = S lCs lna T P = S lTs

In particular embodiments, the oligonucleotide is 10101, 10673, 10674,10677, 10679, 10683, 10707, or 10680, or other oligonucleotide describedin Table 1.

The synthesis of oligonucleotides, including modified polynucleotides,by solid phase synthesis is well known and is reviewed in New ChemicalMethods for Synthesizing Polynucleotides. Caruthers M H, Beaucage S L,Efcavitch J W, Fisher E F, Matteucci M D, Stabinsky Y. Nucleic AcidsSymp. Ser. 1980; (7):215-23.

Compositions, Formulations, and Delivery

The oligonucleotide may be incorporated within a variety ofmacromolecular assemblies or compositions. Such complexes for deliverymay include a variety of liposomes, nanoparticles, and micelles,formulated for delivery to a patient. The complexes may include one ormore fusogenic or lipophilic molecules to initiate cellular membranepenetration. Such molecules are described, for example, in U.S. Pat. No.7,404,969 and U.S. Pat. No. 7,202,227, which are hereby incorporated byreference in their entireties. Alternatively, the oligonucleotide mayfurther comprise a pendant lipophilic group to aid cellular delivery,such as those described in WO 2010/129672, which is hereby incorporatedby reference.

The composition or formulation may employ a plurality of therapeuticoligonucleotides, including at least one described herein. For example,the composition or formulation may employ at least 2, 3, 4, or 5 miRNAinhibitors described herein.

The oligonucleotides of the invention may be formulated as a variety ofpharmaceutical compositions. Pharmaceutical compositions Will beprepared in a form appropriate for the intended application. Generally,this will entail preparing compositions that are essentially free ofpyrogens, as well as other impurities that could be harmful to humans oranimals. Exemplary delivery/formulation systems include colloidaldispersion systems, macromolecule complexes, nanocapsules, microspheres,beads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes. Commercially available fatemulsions that are suitable for delivering the nucleic acids of theinvention to cardiac and skeletal muscle tissues include Intralipid®,Liposyn®, Liposyn® II, Liposyn® III, Nutrilipid, and other similar lipidemulsions. A preferred colloidal system for use as a delivery vehicle invivo is a liposome (i.e., an artificial membrane vesicle). Thepreparation and use of such systems is well known in the art. Exemplaryformulations are also disclosed in U.S. Pat. No. 5,981,505; U.S. Pat.No. 6,217,900; U.S. Pat. No. 6,383,512; U.S. Pat. No. 5,783,565; U.S.Pat. No. 7,202,227; U.S. Pat. No. 6,379,965; U.S. Pat. No. 6,127,170;U.S. Pat. No. 5,837,533; U.S. Pat. No. 6,747,014; and WO03/093449, whichare hereby incorporated by reference in their entireties.

In some embodiments, the oligonucleotide is formulated for conventionalsubcutaneous or intravenous administration, for example, by formulatingwith appropriate aqueous diluent, including sterile water and normalsaline.

The pharmaceutical compositions and formulations may employ appropriatesalts and buffers to render delivery vehicles stable and allow foruptake by target cells. Aqueous compositions of the present inventioncomprise an effective amount of the delivery vehicle comprising theinhibitor oligonucleotide (e.g. liposomes or other complexes), dissolvedor dispersed in a pharmaceutically acceptable carrier or aqueous medium.The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refers to molecular entities and compositions that do notproduce adverse, allergic, or other untoward reactions when administeredto an animal or a human. As used herein, “pharmaceutically acceptablecarrier” may include one or more solvents, buffers, solutions,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like acceptable for usein formulating pharmaceuticals, such as pharmaceuticals suitable foradministration to humans. The use of such media and agents forpharmaceutically active substances is well known in the art.Supplementary active ingredients also can be incorporated into thecompositions.

Administration or delivery of the pharmaceutical compositions accordingto the present invention may be via any route so long as the targettissue is available via that route. For example, administration may beby intradermal, subcutaneous, intramuscular, intraperitoneal orintravenous injection, or by direct injection into target tissue (e.g.,cardiac tissue). The stability and/or potency of the oligonucleotidesdisclosed herein allows for convenient routes of administration,including subcutaneous, intradermal, and intramuscular. Pharmaceuticalcompositions comprising miRNA inhibitors may also be administered bycatheter systems or systems that isolate coronary circulation fordelivering therapeutic agents to the heart. Various catheter systems fordelivering therapeutic agents to the heart and coronary vasculature areknown in the art. Some non-limiting examples of catheter-based deliverymethods or coronary isolation methods suitable for use in the presentinvention are disclosed in U.S. Pat. No. 6,416,510; U.S. Pat. No.6,716,196; U.S. Pat. No. 6,953,466, WO 2005/082440, WO 2006/089340, U.S.Patent Publication No. 2007/0203445, U.S. Patent Publication No.2006/0148742, and U.S. Patent Publication No. 2007/0060907, which areall hereby incorporated by reference in their entireties.

The compositions or formulations may also be administered parenterallyor intraperitoneally. By way of illustration, solutions of theconjugates as free base or pharmacologically acceptable salts can beprepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations generallycontain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use or catheterdelivery include, for example, sterile aqueous solutions or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersions. Generally, these preparations aresterile and fluid to the extent that easy injectability exists.Preparations should be stable under the conditions of manufacture andstorage and should be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. Appropriate solvents ordispersion media may contain, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), suitable mixtures thereof, and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialan antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating theconjugates in an appropriate amount into a solvent along with any otheringredients (for example as enumerated above) as desired. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the desired other ingredients, e.g., as enumerated above. Inthe case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation include vacuum-dryingand freeze-drying techniques which yield a powder of the activeingredient(s) plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Upon formulation, solutions are preferably administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations may easily be administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution generally is suitably buffered andthe liquid diluent first rendered isotonic for example with sufficientsaline or glucose. Such aqueous solutions may be used, for example, forintravenous, intramuscular, subcutaneous and intraperitonealadministration. Preferably, sterile aqueous media are employed as isknown to those of skill in the art, particularly in light of the presentdisclosure. By way of illustration, a single dose may be dissolved in 1ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards:

Methods of Treatment

The invention provides a method for delivering oligonucleotides to amammalian cell (e.g., as part of a composition or formulation describedherein), and methods for treating, ameliorating, or preventing theprogression of a condition in a mammalian patient. The oligonucleotideor pharmaceutical composition may be contacted in vitro or in vivo witha target cell (e.g., a mammalian cell). The cell may be a heart cell.

The method generally comprises administering the oligonucleotide orcomposition comprising the same to a mammalian patient or population oftarget cells. The oligonucleotide, as already described, is a miRNAinhibitor (e.g., having a nucleotide sequence designed to inhibitexpression or activity of a miR-208 family miRNA). Thus, the patient mayhave a condition associated with, mediated by, or resulting from,miR-208 family expression. Such conditions include, for example, cardiachypertrophy, myocardial infarction, heart failure (e.g., congestiveheart failure), vascular damage, restenosis, or pathologic cardiacfibrosis. Thus, the invention provides a use of the modifiedoligonucleotides and compositions of the invention for treating suchconditions, and for the preparation of medicaments for such treatments.

In certain embodiments, the patient (e.g., human patient) has one ormore risk factors including, for example, long standing uncontrolledhypertension, uncorrected valvular disease, chronic angina, recentmyocardial infarction, congestive heart failure, congenitalpredisposition to heart disease and pathological hypertrophy.Alternatively or in addition, the patient may have been diagnosed ashaving a genetic predisposition to, for example, cardiac hypertrophy, ormay have a familial history of for example, cardiac hypertrophy.

In this aspect, the present invention may provide for an improvedexercise tolerance, reduced hospitalization, better quality of life,decreased morbidity, and/or decreased mortality in a patient with heartfailure or cardiac hypertrophy.

In certain embodiments, the activity of miR-208a, miR-208b, and/ormiR-499 in cardiac tissue, or as determined in patient serum, is reducedor inhibited.

In various embodiments, the pharmaceutical composition is administeredby parenteral administration or by direct injection into heart tissue.The parenteral administration may be intravenous, subcutaneous, orintramuscular. In some embodiments, the composition is administered byoral, transdermal, sustained release, controlled release, delayedrelease, suppository, catheter, or sublingual administration. In certainembodiments, the oligonucleotide is administered at a dose of 25 mg/kgor less, or a dose of 10 mg/kg or less, or a dose of 5 mg/kg or less. Inthese embodiments, the oligonucleotide or composition may beadministered by intramuscular or subcutaneous injection, orintravenously.

In some embodiments, the methods further comprise scavenging or clearingthe miRNA inhibitors following treatment. For example, a oligonucleotidehaving a nucleotide sequence that is complementary to the inhibitor maybe administered after therapy to attenuate or stop the function of theinhibitor.

EXAMPLES Example 1 In Vitro Activity of miRNA Inhibitors Targeting themiRNA 208 Family

A panel of miRNA inhibitors (single stranded oligonucleotides) wassynthesized targeting the miRNA 208 family (miR208a, miR-208b andmiR-499). The sequences and modification patterns are shown in Table 1.A description of the base codes is provided in Table 2. The panelincluded multiple lengths of reverse complement inhibitors ranging from11 nucleotides to 16 nucleotides. The number of LNA modifications wasvaried as well as the location of the LNA modification in theoligonucleotide.

A small panel was initially tested in HeLa cells utilizing thedual-luciferase assay readout. The assay used the psiCHECK™-2 construct(Promega) (FIG. 1). HeLa cells do not express the miR-208 family;therefore the corresponding mimic was also co-transfected with theplasmid.

The results show that LNA patterns have disparate activities in vitro asinhibitors of miR-208 family miRNAs. Some particularly potent designsare shown in FIG. 2. For example, M-10673 has the same number of INAmodifications (9 out of 16) as M-10101, yet at 1 nM showed higherinhibition of miR-208a. In view of these results, another limited panelof inhibitors was synthesized and tested, with all being 16 nucleotidesin length with 9 LNA modifications (the remaining being DNAnucleosides). FIGS. 3-5 show the results of these inhibitors in dualluciferase reports for miR-208a, miR-208b, and miR-499 (respectively).M-10673 showed inhibition for not just miR-208a, but also againstmiR-208b and miR-499. There are two mismatches between 208a and miR-208bin the 16-mer inhibitors.

A more complete panel of inhibitor designs was then constructed. Thestructure of these molecules are shown in Table 1.

Example 2 In Vivo Activity of miRNA Inhibitors Targeting the miRNA 208Family

Three inhibitors targeting miR-208 family were synthesized and tested innormal mice for the effect on miR-208a and miR-208b levels. The mice(n=4) were dosed 2.5, 10 and 25 mg/kg through a low pressure tail veininjection and heart tissue was analyzed four days later by qPCR formiRNA levels. The results (FIG. 6) correlated well to the in vitro dualluciferase results. These results suggest that it may be possible tolower the dose at least 10-fold for a therapeutic effect (25 mpk to 2.5mpk).

The above initial experiments demonstrate that there are uniqueLNA-containing modification motifs (including number and position ofLNA) that enhance potency for miR-208 family miRNAs.

M-10101 and M-10673 were tested in the Dahl salt-sensitive rat model,which is described further below. FIGS. 7 and 8 show the best survivaland body weight control with M-10101.

Example 3 Therapeutic Inhibition of miR-208 Improves Cardiac Functionand Survival During Heart Failure

Previously, it was, reported that genetic deletion of the cardiacspecific miR-208a prevents pathological cardiac remodeling andup-regulation of Myh7 in response to stress. This example shows thatsystemic delivery of an antisense oligonucleotide (M-10101 from Table 1)induces potent and sustained silencing of miR-208a in the heart.Therapeutic inhibition of miR-208a by subcutaneous delivery ofantimiR-208a during hypertension-induced heart failure in Dah1hypertensive rats dose-dependently prevents pathological myosinswitching and cardiac remodeling, while improving cardiac function,overall health and survival. Transcriptional profiling indicatesantimiR-208a evokes prominent effects on cardiac gene expression, whileplasma analysis indicates significant changes in circulating levels ofmiRNAs upon antimiR-208a treatment. These studies indicate the potentialof oligonucleotide-based therapies for modulating cardiac miRNAs, andvalidate miR-208 as a potent therapeutic target for the manipulation ofcardiac function and remodeling during heart disease.

Chronic and acute stress to the heart results in a pathologicalremodeling response accompanied by cardiomyocyte hypertrophy, fibrosis,pump failure, myocyte degeneration and apoptosis, which often culminatein heart failure and sudden death (1). While classical pharmacologicaltreatment strategies can reduce remodeling and prolong survival in heartfailure patients, these therapies are ultimately ineffective inpreventing progression of the disease. A hallmark of pathologicalhypertrophy and heart failure is the re-activation of a set of fetalcardiac genes, including those encoding atrial natriuretic factor (ANF),B-type natriuretic peptide (BNP) and fetal isoforms of contractileproteins, such as skeletal α-actin and Myh7 (β-myosin heavy chain,β-MHC) (2). Down-regulation of Myh6 (α-MHC) and up-regulation of Myh7 isa common response to cardiac injury irrespective of the species (3-5).Relatively minor increases in the ratio of Myh6 to Myh7 have been shownto have beneficial effects on cardiac contractility and performance inhumans and rodents (6-8). Much attention has been focused onunderstanding the mechanisms that regulate cardiac remodeling and myosinswitching in search for potential approaches to therapeuticallymanipulate these processes.

Previously, signature expression patterns of microRNAs (miRNAs) wereidentified that were associated with pathological cardiac hypertrophy,heart failure and myocardial infarction in humans and mouse models ofheart disease (9-10). Gain- and loss-of-function studies in mice haverevealed profound and unexpected functions for these miRNAs in numerousfacets of cardiac biology, including the control of myocyte growth,contractility, fibrosis, and angiogenesis (reviewed in 11). Especiallyintriguing is miR-208, a miRNA encoded within an intron of the Myh6 genewhich regulates the cardiac stress response (12-13). Although geneticdeletion of miR-208 in mice failed to induce an overt phenotype atbaseline, in response to several forms of cardiac stress, miR-208 nullmice showed virtually no cardiomyocyte hypertrophy or fibrosis and wereunable to up-regulate Myh7 expression (12).

In the adult heart, miR-208 is essential for the expression of not onlyMyh7, but also of a closely related myosin isoform, Myh7b (14).Remarkably, both of these genes encode slow myosins and contain intronicmiRNAs (miR-208b and miR-499, respectively) (15-16). Since miR-208(which we will refer to as miR-208a), -208b and miR-499 are relatedmiRNAs that arise from myosin genes, we collectively refer to as myomiRs(17). Through gain- and loss-of-function experiments in mice, we haveshown that genetic deletion of miR-208a dose-dependently reducesMyh7b/miR-499 expression within the adult heart (18). Since miR-499mutant animals show no effect on Myh7 expression or cardiac remodelingin response to stress, and reintroduction of miR-499 removes the cardiaceffects seen in the miR-208a mutant mice (18), we conclude that thecombined reduction in miR-208a and miR-499 is responsible for thecardioprotective effects seen in miR-208a mutant animals.

The importance of miRNAs for cardiac function and dysfunction suggestsopportunities for therapeutically exploiting the biology of miRNAs inthe setting of heart disease. Single-stranded RNA oligonucleotides havebeen shown to be effective in inactivating miRNAs in vivo throughcomplementary base pairing (19-23), and represent a potentiallyeffective means of inactivating pathological miRNAs. Here we show thatsystemic delivery of unconjugated, Locked Nucleic Acid (LNA)-modifiedantisense oligonucleotides against miR-208a is sufficient to inducespecific, potent and sustained silencing of miR-208a in the heart.Moreover, antimiR-208a dose-dependently prevents stress-inducedremodeling, functional deterioration, and cardiac myosin switching,while improving general health and survival in a rat model of heartfailure (Dahl salt-sensitive rats). Gene expression analysis showedspecific gene expression changes in response to antimiR-208a treatment,including changes in previously defined target genes. Intriguingly,these physiological effects of antimiR-208a in hypertensive rats aremirrored by significant changes in plasma levels of circulating miRNAs.Together, these studies indicate the potency of systemically deliveredantimiRs in the settings of heart disease, and validate miR208 as animportant therapeutic target during heart failure.

AntimiR Mediated Silencing of miR-208a In Vivo

To determine the therapeutic potential of miR-208a inhibition incardiomyocytes in vivo, we designed an unconjugated LNA-containingantimiR against miR-208a (antimiR-208a, M-10101 in Table 2).AntimiR-208a targets bases 2-17 of the 5′ region of mature mild-208a,and contains a combination of LNA and DNA linked by phosphorothioatebonds. Real-time PCR and Northern blot analysis one week afterintravenous (i.v.) delivery of antimiR-208a to mice at doses rangingfrom 0.1 to 33 mg/kg indicated a dose-responsive silencing of miR-208a,while injection of a mismatch antimiR of similar chemistry showed noinhibition of miR-208a (FIG. 12). Notably, we observed an up-shift ofmiR-208 in the presence of the 16 mer LNA antimiR, reflecting theformation of a stable heteroduplex between miR-208a and the LNA antimiR.Real-time analysis of the other two myomiRs, miR-208b and miR-499,showed no inhibition following a single injection after seven days, nordid we observe any changes in Myh7 (data not shown).

To investigate the potential to deliver antimiR-208a via additionalroutes of administration, we injected mice i.v., intraperitoneally(i.p.), or subcutaneously (s.c.) with 25 mg/kg antimiR-208a and measuredmiR-208a inhibition at days 1, 4, 7, and 14. All 3 routes ofadministration showed robust inhibition of miR-208a (FIG. 13), with nosignificant differences in antimiR-208a detection in plasma, heart,liver and kidney between the different delivery methods (not shown).

Extended miR-208a Inhibition Leads to Myh7 Regulation In Vivo

Since a single dose of antimiR-208a after seven days was unable toestablish an effect on Myh7, as was seen in the miR-208a knockout mice,we set out to determine the dose and time required for efficient Myh7regulation following antimiR-208a administration. Three consecutivedoses of 33 mg/kg antimiR-208a robustly inhibited miR-208a for at leastsix weeks (FIG. 14). miR-499, which is known to be regulated by miR-208(18), showed a time-dependent decrease in expression from one tosix-weeks after administration of antimiR-208a, going from a 35 to 75%reduction in miR-499 (FIG. 14A). Furthermore, Myh7 mRNA expression wassignificantly reduced starting at four weeks after antimiR-208atreatment, suggesting a specific threshold of miR-208a and miR-499levels is necessary for Myh7 expression (FIG. 14B), which was paralleledby a reduction in Myh7 protein (FIG. 14C). The initial spike in Myh7mRNA in response to antimiR-208a is not translated into increased Myh7protein.

To establish whether the effect on Myh7 expression is based on areduction in both miR-208a and miR-499, we injected mice for 3consecutive days with a cocktail of antimiR-208a and antimiR-499, eachat 33 mg/kg. Treatment with antimiR-208a/-499 caused robust inhibitionof miR-208a and miR-499 for six weeks, and demonstrated a much morerapid regulation of Myh7 mRNA and protein, with reduced expressionbefore two weeks after treatment (FIG. 14A-C). AntimiR distribution datausing a sandwich hybridization assay to quantify antimiR-208a in heart,liver, kidney, and plasma, indicated that considerable amounts ofantimiR-208a are still detectable 6 weeks after administration of either33 mg/kg or 3×33 mg/kg of antimiR-208a (FIG. 15)

Therapeutic Silencing of miR-208 Reduces Cardiac Remodeling, whileImproving Cardiac Function and Survival During Heart Failure

Since previous data showed that genetic deletion of miR-208a results ina cardioprotective effect, we aimed to test the therapeutic relevance ofmiR-208a To this end, we used Dahl salt-sensitive rats that were eitherfed a low-salt (LS) diet (0.25% NaCl) or a high-salt (HS) diet (8.0%NaCl) starting at 8 weeks of age. After one week on HS, rats wereadministered saline, 25 mg/kg antimiR-208a, or 25 mg/kg scrambledcontrol oligo subcutaneously every two weeks. Following 3-4 weeks on theHS diet, the saline and control treated animals showed visible signs ofimmobility and discomfort and death, while subcutaneous delivery ofantimiR-208a was able to significantly alleviate these symptoms (FIG.16). As an indication of health, we monitored body weight during theduration of the study. Dahl rats on the HS diet injected with eithersaline or the control oligo exhibited significant reductions in weightgain compared to LS diet controls. HS/antimiR-208a treated rats,however, showed comparable weight gain (FIG. 16B). To exclude thepossibility antimiR-208a treated animals were maintaining weight throughingesting less of the 8% HS diet, food intake was monitored, whichshowed a comparable ingestion between all HS fed groups (not shown).

To obtain additional insight into the protective effects seen inresponse to antimiR-208a, subsequent studies were done using a 4.0% NaCldiet for 9 weeks, during which the rats received either saline, 5 or 25mg/kg of antimiR-208a, or 25 mg/kg of antimiR control every 2 weeks.Body weight analysis indicated that Dahl rats on the HS diet exhibitedsignificant reductions in weight gain compared to LS diet controls,while HS/antimiR-208a treated rats maintained their increase in weightgain (FIG. 17A). Functional assessment using echocardiography ofantimiR-208a treated Dahl rats showed a dose-dependent, significantimprovement in measurements of diastolic function. AntimiR-208a treatedrats exhibited a significant reduction in isovolumic relaxation time(IVRT) compared to HS/saline controls, as well as a normalization of themitral valve early to active filling velocity ratio (MV E/A) compared toHS/Saline controls eight weeks post HS diet (FIG. 17B). Quantificationof cardiomyocyte size showed a significant reduction in cardiomyocytehypertrophy following treatment with antimiR-208a (FIG. 18A, B).Additionally, antimiR-208a treatment reduced periarteriolar fibrosisinduced by HS diet as assessed by quantification of picrosirius redstaining (FIG. 18A, B).

miR-208a Inhibition Reverses the Myosin Switch During Heart Failure

To compare the physiological changes observed after antimiR-208atreatment with molecular and cellular changes, we examined myomiRexpression following HS treatment. AntimiR-208a caused a dose-dependentinhibition of miR-208a in both left and right ventricles 2 weeks afterthe last injection, whereas a control oligo showed no differencecompared to saline (FIG. 19A, left panel). miR-499 also showed adose-dependent decrease in expression following sustained inhibition ofmiR-208a (FIG. 19A, middle panel). miR-208b was induced in bothHS/Saline and HS/Control treated animals, however antimiR-208a treatmentresulted in a dose-dependent decrease in miR-208b levels (FIG. 19A,right panel). This regulation of miR-499 and miR-208b was confirmed byNorthern blot analysis (FIG. 19B).

To assess the regulation of the host genes, we examined Myh6, Myh7, andMyh7b mRNA levels. Myh7 was significantly increased in response to HS inboth the HS/saline and HS/control groups. This increase wasdose-dependently blunted in response to antimiR-208a. Additionally,antimiR-208a treatment normalized the decreased expression of Myh6 mRNAobserved in both HS/saline and HS/control groups (FIG. 20A). Expressionof Myh7b mirrored miR-499 levels, exhibiting a dose-dependent reductionupon antimiR-208a treatment. Furthermore, the dose-dependent regulationof Myh7 was confirmed by western blot (FIG. 20B).

AntimiR-208a does not Induce Changes in Cardiac Conductance or Signs ofToxicity

While genetic deletion of miR-208a does not affect viability or causegross morphological heart defects, a previous report mentioned thatmiR-208a might be required for proper heart electrophysiology. Althoughwe never observed any overt abnormalities in the miR-208 knockoutanimals, to verify whether antimiR-208a treatment resulted in cardiacconductance effects we measured ECGs in both wild-type mice and diseasedrats. Both species showed proper cardiac electrophysiology afterantimiR-208a treatment for an extended period of time (not shown).

Independent of the route of administration, all mice and rats toleratedthe antimiR-208a or control oligo well and exhibited normal behaviors,as determined by activity level and grooming throughout the study.Compared to saline, antimiR-208a or the control oligo did not inducebaseline changes in body or additional tissue weights, including heart,kidney, liver, lungs or spleen up to 6 weeks after dosing (not shown).Neither antimiR-208a, nor control oligo treatment changed serum levelsof the alanine aminotransferase (ALT) and aspartate aminotransferase(AST) liver enzymes in rats (not shown), suggesting that theoligonucleotides do not induce any overt liver toxicities.

AntimiR-208a Induces Specific Gene Expression Changes

To establish the effect of miR-208a inhibition on gene expressionchanges, we performed microarray analysis on Dahl rats on HS diet thatwere either injected with saline, antimiR-208a or control oligo.Compared to control oligo treated animals, antimiR-208a treated animalsshowed that 131 genes were significantly changed. Only 15 genes (with afalse positive discovery rate of 67%) were significantly differentbetween saline and control oligo injected animals, indicating the lackof effect on gene expression by the oligonucleotide chemistry itself. Asvisually demonstrated in a heat map, hierarchical clustering of theexpression of the 131 significantly changed genes between control oligoand antimiR-208a treated hearts showed robust clustering of up- anddown-regulated genes following antimiR-208a treatment, and validatedthere to be no gene expression response following control oligotreatment (not shown). Gene array analysis confirmed the significantdown-regulation of Myh7 and Myh7b in response to antimiR-208a comparedto control oligo (−1.31, p=0.005 and −2.38, p=0.037, respectively),while Thrap1, a previously characterized target, was increased (1.56,p=0.49). Out of the 13518 genes that were detected on the array, 289genes were bioinformatically predicted to be miR-208 targets. Of thesepredicted targets, 28 genes showed increased expression with antimiR-208treatment by microarray, of which several were confirmed by real-timePCR (not shown). Since the gene expression analysis was performed oncardiac samples from Dahl hypertensive rats that had been treated withsaline, antimiR-208a or control oligo for 8 weeks, we suspect theremainder of the gene expression changes might be secondary to thedirect gene regulatory effects of miR-208a inhibition.

BLAST analysis of the antimiR sequence against the rat genome indicatedthat the sequence of antimiR-208a Shows close homology (at least 14bases of complementarity) to four coding sequences; however none ofthese genes were regulated as determined by microarray analysis.Together these analyses indicate that the LNA-modified oligos are highlyspecific in targeting miR-208a without any gene expression changesinduced by the chemistry class.

miR-499 is Plasma Biomarker for antimiR-208a Efficacy

Detection of miRNAs in plasma during various disease settings is showingincreasing diagnostic promise. To determine if there is a specific miRNAto correlate with antimiR-208a efficacy, we examined a panel of musclerelated miRNAs during HS treatment. Several muscle specific miRNAstested, such as miR-1 and -133, did not show significant differencesbetween the groups tested (not shown). Strikingly, miR-499, while onlyshowing modest increases in plasma detection under high salt, wassignificantly reduced in antimiR-208a treated animals, suggestingmiR-499 can act as a plasma based marker for antimiR-208a efficacy.Additionally, miR-423-5p, plasma levels of which were previouslycorrelated to human heart failure (24), was found to be reduced inanimals treated with antimiR-208a.

Discussion

Data presented here indicate that therapeutic inhibition of miR-208leads to a profound reduction in cardiac remodeling, which coincideswith a significant improvement in survival and cardiac function duringheart disease.

Antisense oligonucleotides can be used to effectively silence miRNAs invivo (19-23). These antimiRs are chemically modified to ensure in vivostability, specificity and high binding affinity to the miRNA ofinterest. LNA is a nucleic acid modification that introduces athermodynamically strong duplex formation with oligonucleotides whileenhancing specificity toward complementary RNA or DNA oligonucleotides(19-20). As a consequence of the high binding affinity, biologicalactivity for LNA-modified antimiRs is attained with shorteroligonucleotides (8-16 bases) (25). Recently, the therapeuticapplicability has been reported in rodents and non-human primates, wheresystemic delivery of unconjugated LNA-antimiR potently antagonized theliver-expressed miR-122 leading to an improvement in Hepatitis CVirus-induced liver pathology in chronically infected chimpanzees (23).

A key finding in the current study is that systemic delivery ofLNA-modified oligonucleotides is effective in inducing potent andsustained silencing of miR-208 in the heart. Sustained miR-208ainhibition and the absence of an effect on the closely related miR-208bupon systemic delivery of antimiR-208a indicate in vivo stability andspecificity. Based on the sustained miR-208a silencing and thedownstream Myh7 regulation in time, it seems probable that antimiR-208acan accumulate in cardiac cells to silence all newly formed copies ofmiR-208a that are being produced by Myh6 transcription. This effectmight be reinforced further by the general lack of turnover ofcardiomyocytes, preventing dilution due to a decrease in the portion ofcells that are targeted with the antimiR.

Although gene regulatory effects of miRNAs on direct targets are fairlyimmediate, miR-208a inhibition requires several weeks before itestablishes an effect on Myh7b and Myh7 expression. We hypothesize thatthe delay in downstream biological effects is due to the requirement ofalterations in the expression of many direct and indirect target genesof which the combined effects are required to induce the change. Acomparable phenomenon was observed in response to miR-122 inhibition,which induces a lowering in plasma cholesterol, but not until weeksafter antimiR treatment while gene expression changes were immediate(19-20). Nonetheless, the effect on Myh7b and Myh7 expressionphenocopies the effects seen in the miR-208 genetic deletion (12),indicating miR-208a is effectively silenced.

The therapeutic effects of antimiR-208a in the Dahl hypertensive ratprovide strong evidence that subcutaneous delivery is sufficient toeffectively deliver antimiRs to the heart in vivo and that miR-208ainhibition prevents cardiac remodeling, functional deterioration andlethality during heart disease. Although it remains unclear whetherthese effects arise solely from effects on the cardiomyocyte due tomiR-208a inhibition, or whether there are extra-cardiac effects inresponse to miR-208a inhibition currently unknown, thedose-responsiveness and the absence of an effect in animals treated witha control chemistry strongly suggest the observed effects are due to alowering in miR-208a levels. Ongoing experiments will indicate whetherthis therapeutic benefit can be established in multiple models of heartfailure and whether combined antimiR dosing against miR-208a and miR-499in parallel will elucidate the observed effects more rapidly. While theinitial rodent data look very encouraging and no adverse side-effectswere observed upon antimiR treatment, extensive analyses will berequired to determine the long-term safety of such agents in varioussettings.

Recently, miRNAs were detected in serum and plasma of humans andanimals, opening the possibility of using miRNAs as diagnosticbiomarkers of various diseases, including heart disease (24, 26-28).Plasma miRNA analysis shows that, in addition to several other miRNAschanging, antimiR-208a treatment results in a diminished detection ofmiR-499 in blood serum, which parallels the decrease in cardiacexpression of Myh7b/miR-499 in response to antimiR-208a treatment. Giventhe correlation between cardiac and plasma based miR-499 levels andefficacy of antimiR-208a, these data suggest plasma miR-499 levels mightact as a biomarker of effective delivery of antimiR-208a to the heartwhen moving into patients.

Myosin and subsequent myomiR expression differs significantly betweenspecies. While Myh6/miR-208a is the predominant myosin/myomiR isoform inthe hearts of smaller rodents, larger mammals express more Myh7/miR208b(17). While miR-208a and 208b have overlapping seed sequence, theydiffer 3 bases in their 3′ region. Subsequent pharmacokinetic andefficacy studies in larger mammals will be required to establish whetherinhibition of miR-208a, miR-208b or both miR-208 isoforms is required toestablish a comparable therapeutic effect in larger species.Additionally, since therapeutic use of miR-208 inhibition will likely bea combination therapy with current standard of care in heart failurepatients, it will be important to assess whether antimiR-208a, inconjunction with these current treatments, adds to the beneficialeffects of these drugs.

Taken together, this study demonstrates that subcutaneous delivery ofLNA-based antimiRs can effectively target the heart, and furthervalidates miR-208 as a target during cardiac disease.

Methods

Animal procedures. All animal protocols were approved by theinstitutional Animal Care and Use Committee of miRagen Therapeutics,Inc.

Animals and Delivery of LNA-Modified antimiRs.

The LNA-antimiR oligonucleotides were synthesized at miRagenTherapeutics, Inc. as unconjugated and fully phosphorothiolatedoligonucleotides perfectly complementary to the 5′ region of the maturemiR-208a sequence. The LNA control oligonucleotide consisted of asequence directed against a C. elegans specific miRNA. Unless elseindicated, in vivo delivery of the oligonucleotide chemistries wasachieved by low pressure intravenous (i.v.) injections via the tail veinof either adult male C56B16 mice or adult male Dahl Salt-sensitive rats(Harlan, Indianapolis). All chemistries were dissolved and injected in acomparable end volume of saline after which the animals were examinedfor obvious side effects of the chemistries. Tissue samples werecollected at the indicated timepoints for molecular or histologicalexamination. Dahl rats were maintained on 0.25 NaCl or placed on 4% or8% NaCl diet at 8 weeks of age (Harlan, Indianapolis).

Quantitative Real-Time PCR Analysis.

For in vivo real-time PCR analysis, RNA was extracted from cardiactissue using Trizol (Invitrogen) after which two μg RNA from each tissuesample was used to generate cDNA using Super Script II reversetranscriptase per manufacturer's specifications (Invitrogen). To detectthe level of miR-208 RT-PCR was performed using the Taqman MicroRNAassay (Applied Biosystems, ABI) according the manufacturer'srecommendations, using 10-100 ng of total RNA. The expression of asubset of genes was analyzed by quantitative real time PCR using Taqmanprobes purchased from ABI.

Northern Blot Analysis.

Total RNA was isolated from cardiac tissue samples by using Trizolreagent (Gibco/BRL). Northern blots to detect microRNAs were performedas described previously described. A U6 probe served as a loadingcontrol (IDT). 10 ug of total RNA from cardiomyocytes or heart tissuewas loaded on 20% acrylamide denaturing gels and transferred toZeta-probe GT genomic blotting membranes (Bio-Rad) by electrophoresis.After transfer, the blots were cross-linked and baked at 80° C. for 1hr. To maximize the sensitivity of miRNA detection, oligonucleotideprobes were labeled with the Starfire Oligos Kit (IDT, Coralville, Iowa)and α-³²P dATP (Amersham or Perkin Elmer). Probes were hybridized to themembranes overnight at 39° C. in Rapid-hyb buffer (Amersham), afterwhich they were washed twice for 10 minutes at 39° C. with 0.5×SSCcontaining 0.1% SDS. The blots were exposed and quantified byPhosphorImager analysis (GE HealthCare Life Sciences) and a U6 probeserved as a loading control (ABI). The intensity of the radioactivesignal was used to quantify the fold change in expression using aphosphorimager and ImageQuant (Bio-Rad).

Western blot analysis. For Western blot analysis, Myosin was extractedfrom cardiac cells or tissue as described (29). MHC isoforms weredetected by loading 0.1 ug protein lysate on a 4-15% gradient gel andseparated by SDS PAGE and Western blotting was performed with mousemonoclonal anti-myosin (slow, skeletal M8421) (Sigma, Mo.), which ishighly specific for Myh7.

Biodistribution Assay.

A sandwich hybridization assay was used for the quantification ofantimiR-208a in plasma and tissue samples. Probes for the hybridizationassay were synthesized using 2′Ome, and LNA modified nucleotides andare: bTEG-mU;1A;mA;1G;mA;1C;mG (capture probe) andmA;1G;mC;1A;mA;1A;mA;1A;mG-6FAM (detection probe). Detection wasaccomplished using anti-fluorescence-POD, Fab fragments (Roche) and TMBPeroxidase Substrate (KPL). Standard curves were generated usingnon-linear logistic regression analysis with 4 parameters (4-PL). Theworking concentration range of the assay was 2-536 ng/ml. Tissue sampleswere prepared at 100 mg/ml by homogenizing in 3M GITC buffer (3 Mguanidine isothiocyanate, 0.5 M NaCl, 0.1 M Tris pH 7.5, 10 mM EDTA) for2×30 seconds using an MP FastPre-24 at a speed setting of 6.0. Plasmasamples and tissue homogenates were diluted a minimum of 50-fold in 1 MGITC Buffer (1 M guanidine isothiocyanate, 0.5 M NaCl, 0.1 M Tris pH7.5, 10 mM EDTA) for testing.

Echocardiography.

Cardiac function was evaluated by two-dimensional transthoracicechocardiography on sedated rats (2-2.5% isoflurane) using a VisualSonic Ultrasound system with a 30 MHz transducer. The heart was imagedin a parasternal short-axis view at the level of the papillary muscles,to record M-mode measurements, determine heart rate, wall thickness, andend-diastolic and end-systolic dimensions. Fractional shortening(defined as the end-diastolic dimension minus the end-systolic dimensionnormalized for the end-diastolic dimension) was used as an index ofcardiac contractile function. Diastolic function was assessed usingtrans-mitral flow Doppler from an apical 4-chamber view to measure E/Aratio, isovolumic relaxation time and deceleration time of E wavevelocity.

Surface ECG Measurement.

Mice were anesthetized with 2% isoflurane in 200 mL/min O₂ and rats wereanesthetized with 2% isoflurane in 500 mL/min breathing air vianosecone. Body temperature for mice and rats was maintained at 37°-38°C. via a Homeothermic Warming System (Kent Scientific) or a heat lampand warming platform (Visual Sonics). Lead II electrocardiograms wererecorded for 10 min using subcutaneous needle electrodes and an Iworxdata acquisition system sampling at 1 kHz. Using Labscribe software(Iworx), tracing were analyzed after 2, 4, 6, 8 and 10 minutes and wereinspected for normal sinus rhythm; approximately 40 beats at eachtimepoint were analyzed using computerized techniques to quantify signalintervals (HR, PR, QRS, QT and QTc).

Histology.

Tissues used for histology were incubated in Krebs-Henselheit solution,fixed in 4% paraformaldehyde, sectioned, and processed for hematoxylinand eosin (H&E) and picrosirius red staining or in situ hybridization bystandard techniques (30). Images of approximately 100 cardiomyocytes peranimal in cross section were captured from the H&E stained sections.Cardiomyocyte cross sectional areas were measured with Image-Pro Plussoftware and a mean was determined for each animal. Perivaseularfibrosis images were taken from epi, mid and endocardial regions fromthe pricrosirius red stained sections from each animal. Image-Pro plussoftware was used to determine the total vessel wall area includingperivascular fibrosis. The luminal area was subtracted from total vesselwall area. Perivascular fibrosis was determined via color segmentationand reported as a % of the total vessel wall area.

Gene expression analysis. Microarray profiling was performed on IlluminaRatRef-12 BeadChip arrays by a service provider (Expression Analysis,Durham, N.C.). Total RNA was isolated from cardiac tissue as describedabove. Analysis of differential gene expression was performed by theservice provider using PADE (Permutation Analysis of DifferentialExpression). Note that if a gene probe does not have detection p-value≦0.05 in all 12 arrays, then that gene is omitted from subsequentanalysis. Differential expression graphs were provided by the serviceprovider. Gene clustering was performed using Cluster 3.0 and heat mapimages were generated in Java TreeView. Gene ontology was performedusing the online tool found at www.pantherdb.org. Predicted miR-208 genetargets in the rat were found using targetscan.org (TargetScan),pictar.mdc-berlin.de (Pictar), and microrna.org (miRanda). Of all thegene targets predicted by miRanda, only those with a mirsvr score of<−0.1 were included in the analysis. For the identification of miR-208targets, a p-value cut-off for differential expression of ≦0.05 wasused.

Quantitative Real-Time PCR Analysis from Plasma.

RNA from plasma samples was isolated using Trizol LS Reagent(Invitrogen), using the manufacturer's protocol. Prior to RNA isolation,250 pmol of two different synthetic C. elegans miRNA sequences wereadded to serve as internal controls for normalization of target miRNAs.The C. elegans sequences used were cel-miR-2 (UAUCACAGCCAGCUUUGAUGUGC(SEQ ID NO:92)), and cel-lin-4 (UCCCUGAGACCUCAAGUGUGA (SEQ ID NO:93))(Dharmacon). The final RNA pellet was re-suspended in a final volumeequal to the initial plasma volume and 5 μl was used for subsequentRT-PCR reactions, as described above.

Statistical Analysis.

One-way ANOVA and Newman-Keuls Multiple Comparison Post-test were usedto determine significance. P<0.05 was considered statisticallysignificant.

Example 4 Inhibitor Dosing in Non-Human Primates

Antimirs 10101 and 10707 were administered three times at a dose of 25mg/kg to African Geen Monkeys (˜3 kg) by the saphenous vein. Tissue wascollected after four weeks and assayed for inhibitor. Results are shownin FIG. 22. Right panel shows drug plasma clearance. Left panel showstissue and plasma distribution (dark bars, M-10101; light bars,M-10707).

FIG. 23 shows miRNA target inhibition. Left panel shows changes inmiR-208a expression in left ventricle (left to right: untreated,M-10101, 10707, 10591). Right panel shows changes in miR-208b in leftventricle (left to right: untreated, M-10101, M-10707, M-10591). Withonly two nucleotide differences between M-10101 and M-10707, theantimiRs are specific for their target miR (miR-208a and miR-208b,respectively).

FIG. 24 shows Mir-499 levels after treatment. Levels are shown for leftventricle (LV), right ventricle (RV), and septum. Bars are, from left toright, untreated, M-10101, M-10707, and M-10591.

Example 5 Molecular Analysis of antimiR-208a Treatment

Seven antimiR-208a chemistries where selected that showed efficacy invivo, each having 9 LNAs and 7 DNA nucleotides. The compounds were dosedat 25 mg/kg s.c. in mice, with takedown at day 4. miRs and targetexpression were measured. Compounds were: M-10101, M-10680, M-10681,M-10682, M-10683, M-10673, and M-11184 (see Table 1).

Hepatic and renal toxicology markers did not show significant increasesfrom saline (data not shown).

The compounds show varying levels of target de-repression. M-10101 andM-10683 were particularly effective. FIG. 25 shows expression ofmiR-208a and Dynlt1. FIG. 26 shows expression of Dynlt1, Vcpip, andTmbim6. FIG. 27 shows expression of Thrap1 and Sp3. FIG. 28 showsexpression of Purb, Gata4, and Sox6.

As shown in FIG. 29, antimir-208a treatment increases miR-19b plasmalevels in unstressed rodents (SD rats).

The degree of target de-repression depends on the degree of stress, asshown using the Dahl salt-sensitive rat model. FIG. 30 shows the resultsfor Dynlt1 expression at 4% salt and 6% salt. Dynlt1 shows more robustde-repression at 6%. FIG. 31 shows results for the target Vcpip1. FIG.32 shows results for the target Tmbim6.

FIGS. 33 to 39 show degrees of miR inhibition in different regions ofthe heart, showing that more stressed regions show greater effect. FIG.33 shows inhibition of miR-208a, miR-208b, and miR-499, FIG. 34 showsde-repression of myosin markers. FIG. 35 shows degree of expression ofcertain cardiac stress markers. FIG. 36 shows de-repression of Dynlt1,Vcpip, Tmbim6 and Cbx1. FIG. 37 shows expression of Thrap1, Sox6, Sp3,and pur-beta. As shown in FIG. 38, the infarted area showed the greatestde-repression of Dynlt1. FIG. 39 shows the de-repression of targets indifferent regions of the heart with M-10101.

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1. An oligonucleotide comprising a nucleotide sequence that issubstantially complementary to a nucleotide sequence of human miR-208aor miR-208b, and having a mix of locked and non-locked nucleotides,wherein the length of the oligonucleotide and number and position oflocked nucleotides is such that the oligonucleotide reduces miR-208a,miR-208b, and/or miR-499 activity at an oligonucleotide concentration ofabout 50 nM or less in an in vitro luciferase assay, or at a dose of 25mg/kg or less in a mouse model. 2-47. (canceled)
 48. A method ofpreventing or treating pathologic cardiac hypertrophy in a subjectassociated with or mediated by miR-208a, miR-208(b), and/or miR-499,comprising administering to the subject a pharmaceutical compositioncomprising an effective amount of an oligonucleotide comprising anucleotide sequence that is substantially complementary to a nucleotidesequence of human miR-208a or miR-208b, and having a mix of locked andnon-locked nucleotides, wherein the length of the oligonucleotide andnumber and position of locked nucleotides is such that theoligonucleotide reduces miR-208a, miR-208b, and/or miR-499 activity atan oligonucleotide concentration of about 50 nM or less in an in vitroluciferase assay, or at a dose of 25 mg/kg or less in a mouse model. 49.A method of preventing or treating myocardial infarction in a subjectassociated with or mediated by miR-208a, miR-208(b), and/or miR-499,comprising administering to the subject a pharmaceutical compositioncomprising an effective amount of an oligonucleotide comprising anucleotide sequence that is substantially complementary to a nucleotidesequence of human miR-208a or miR-208b, and having a mix of locked andnon-locked nucleotides, wherein the length of the oligonucleotide andnumber and position of locked nucleotides is such that theoligonucleotide reduces miR-208a, miR-208b, and/or miR-499 activity atan oligonucleotide concentration of about 50 nM or less in an in vitroluciferase assay, or at a dose of 25 mg/kg or less in a mouse model. 50.A method of preventing or treating heart failure in a subject associatedwith or mediated by miR-208a, miR-208(b), and/or miR-499, comprisingadministering to the subject a pharmaceutical composition comprising aneffective amount of an oligonucleotide comprising a nucleotide sequencethat is substantially complementary to a nucleotide sequence of humanmiR-208a or miR-208b, and having a mix of locked and non-lockednucleotides, wherein the length of the oligonucleotide and number andposition of locked nucleotides is such that the oligonucleotide reducesmiR-208a, miR-208b, and/or miR-499 activity at an oligonucleotideconcentration of about 50 nM or less in an in vitro luciferase assay, orat a dose of 25 mg/kg or less in a mouse model.
 51. A method ofpreventing or treating vascular damage in a subject associated with ormediated by miR-208a, miR-208(b), and/or miR-499, comprisingadministering to the subject a pharmaceutical composition comprising aneffective amount of an oligonucleotide comprising a nucleotide sequencethat is substantially complementary to a nucleotide sequence of humanmiR-208a or miR-208b, and having a mix of locked and non-lockednucleotides, wherein the length of the oligonucleotide and number andposition of locked nucleotides is such that the oligonucleotide reducesmiR-208a, miR-208b, and/or miR-499 activity at an oligonucleotideconcentration of about 50 nM or less in an in vitro luciferase assay, orat a dose of 25 mg/kg or less in a mouse model.
 52. A method ofpreventing or treating restenosis in a subject associated with ormediated by miR-208a, miR-208(b), and/or miR-499, comprisingadministering to the subject a pharmaceutical composition comprising aneffective amount of an oligonucleotide comprising a nucleotide sequencethat is substantially complementary to a nucleotide sequence of humanmiR-208a or miR-208b, and having a mix of locked and non-lockednucleotides, wherein the length of the oligonucleotide and number andposition of locked nucleotides is such that the oligonucleotide reducesmiR-208a, miR-208b, and/or miR-499 activity at an oligonucleotideconcentration of about 50 nM or less in an in vitro luciferase assay, orat a dose of 25 mg/kg or less in a mouse model.
 53. A method ofpreventing or treating pathologic cardiac fibrosis in a subjectassociated with or mediated by miR-208a, miR-208(b), and/or miR-499,comprising administering to the subject a pharmaceutical compositioncomprising an effective amount of an oligonucleotide comprising anucleotide sequence that is substantially complementary to a nucleotidesequence of human miR-208a or miR-208b, and having a mix of locked andnon-locked nucleotides, wherein the length of the oligonucleotide andnumber and position of locked nucleotides is such that theoligonucleotide reduces miR-208a, miR-208b, and/or miR-499 activity atan oligonucleotide concentration of about 50 nM or less in an in vitroluciferase assay, or at a dose of 25 mg/kg or less in a mouse model.